Sorting particles using high gradient magnetic fields

ABSTRACT

This disclosure describes microfluidic devices that include one or more magnets, each magnet being operable to emit a magnetic field; and a magnetizable layer adjacent to the one or more magnets, in which the magnetizable layer is configured to induce a gradient in the magnetic field of at least one of the magnets. For example, the gradient can be at least 10 3  T/m at a position that is at least 20 μm away from a surface of the magnetizable layer. The magnetizable layer includes a first high magnetic permeability material and a low magnetic permeability material arranged adjacent to the high magnetic permeability material. The devices also include a microfluidic channel arranged on a surface of the magnetizable layer, wherein a central longitudinal axis of the microfluidic channel is arranged at an angle to or laterally offset from an interface between the high magnetic permeability material and the low magnetic permeability material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/410,985, filed Dec. 23, 2014, which is a 371 U.S. NationalApplication of PCT Application No. PCT/US2013/047710, filed on Jun. 25,2013, which claims priority to U.S. Provisional Application No.61/664,051, filed on Jun. 25, 2012. The entire contents of the aboveapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to sorting particles using high gradientmagnetic fields.

BACKGROUND

Magnetic cell separation is a technique in which magnetic fields areused to isolate cells within a fluid sample. Typically, magneticparticles are selectively attached to one or more desired cells usingantibodies that bind to the cell surface. Cells having the attachedmagnetic particles then can be confined or deflected using an appliedmagnetic field to isolate the magnetically labeled cells from otheranalytes in the fluid sample.

SUMMARY

In general, one aspect of the present disclosure can be embodied inmicrofluidic devices that employ high magnetic field gradients forsorting particles flowing within a microfluidic channel of the device.The devices can include one or more magnets and a layer having a highmagnetic permeability region surrounding a low magnetic permeabilityregion. The high magnetic permeability region provides a preferred pathfor flux lines emanating from the one or more magnets, such that themagnetic field lines extend over the low magnetic permeability region toestablish a fringing flux field with a high field gradient. The highfield gradient, which is at least about 10³ T/m at a distance of atleast 20 μm from the channel floor, then can be used to establish amagnetic force on magnetic particles flowing within the adjacentmicrofluidic channel for sorting.

In accordance with a general aspect 1 of the disclosure, microfluidicdevices are provided that include one or more magnets, each magnet beingoperable to emit a magnetic field; a magnetizable layer adjacent to theone or more magnets, in which the magnetizable layer is configured toinduce a gradient in the magnetic field of at least one of the magnets,the gradient being at least 10³ T/m at a position that is at least 20 μmaway from a surface of the magnetizable layer, and in which themagnetizable layer includes a first high magnetic permeability material,and a low magnetic permeability material arranged adjacent to the highmagnetic permeability material; and a microfluidic channel arranged on asurface of the magnetizable layer, wherein a central longitudinal axisof the microfluidic channel is arranged at an angle to or laterallyoffset from an interface between the high magnetic permeability materialand the low magnetic permeability material.

The foregoing and other embodiments can each optionally include one ormore of the following features, alone or in combination. For example, inaspect 2 according to aspect 1, the high magnetic permeability materialand the low magnetic permeability material are elongated. In aspect 3,according to any one of aspect 2 to 3, the device further includes asecond high magnetic permeability material arranged at a distance fromthe first high magnetic permeability material to form a gap between them(e.g., a uniform gap), wherein the gap is filled with low magneticpermeability material. In aspect 4, according to aspect 3, the lowmagnetic permeability material in the gap is the same as the lowmagnetic permeability material in a remainder of the magnetizable layer.

In aspect 5, according to any one of aspects 1 to 4, the gradient can beat least 10⁴ T/m at a position of at least 20 μm away from the surfaceof the magnetizable layer, e.g., at least 10⁴ T/m at a position of atleast 50 μm away from the surface of the magnetizable layer.

In aspect 6, according to any one of aspects 1 to 5, a thickness of thehigh magnetic permeability material is greater than or equal toapproximately 10 μm, e.g., greater than or equal to approximately 100μm, or greater than or equal to approximately 1 mm.

In aspect 7, according to any one of aspects 1 to 6, a saturation fluxdensity of the high magnetic permeability material is greater than 1 T,e.g., greater than 1.5 T. In aspect 8, according to any one of aspects 1to 6, the saturation flux density can be about 1.8 T.

In aspect 9, according to any one of aspects 1 to 8, the microfluidicdevices include a low magnetic permeability spacer layer between the oneor more magnets and the magnetizable layer.

In aspect 10, according to any one of aspects 3 to 9, a thickness of thegap is equal to or less than a thickness of each of the first and secondhigh magnetic permeability material.

In aspect 11, according to any one of aspects 1 to 10, the angle is anoblique angle, e.g., an acute angle, such as an angle of from about 0.5°to about 30°, e.g., 1°, 2°, 3°, 5°, 10, 15°, 20°, or 25° measured from alongitudinal axis of the microfluidic channel of the device.

In aspect 12, according to any one of aspects 1 to 11, the high magneticpermeability material includes iron. In aspect 13, according to any oneof aspects 1 to 12, the high magnetic permeability material is selectedfrom the group including or consisting of iron, nickel, cobalt,Nickel-Iron alloy, SiFe alloy, FeAlN alloy, a CoFe alloy, CoFeNi, steel,a polymer composite containing magnetic particles, a glass compositecontaining magnetic particles, and a ceramic composite containingmagnetic particles.

In aspect 14, according to any one of aspects 1 to 13, the microfluidicdevice includes an elongated cover defining a top surface and sidesurfaces of the microfluidic channel.

In aspect 15, according to any one of aspects 1 to 14, the low magneticpermeability material can be a non-magnetic material such as a polymeror air.

In aspect 16, according to any one of aspects 1 to 15, the microfluidicdevice includes a passivation layer arranged on the surface of themagnetizable layer and forming a bottom surface of the microfluidicchannel.

In aspect 17, according to any one of aspects 1 to 16, a width of thelow magnetic permeability material can narrow from a first side of themagnetizable layer to a second opposite side of the magnetizable layer.

In aspect 18, according to any one of aspects 1 to 17, the microfluidicdevices include an array of two or more magnets, each magnet having amagnetic pole orientation that is opposite to a magnetic poleorientation of an adjacent magnet in the array such that the magneticfield of each magnet in the array extends to an adjacent magnet in thearray. In aspect 19, according to aspect 18, the elongated low magneticpermeability material can be substantially aligned with an interfacebetween two adjacent magnets of the array.

In aspect 20, according to aspects 1 to 19, the microfluidic devicesinclude a deflection channel and, an output channel separate from thedeflection channel, in which both the output channel and the deflectionchannel are fluidly coupled to an outlet of the microfluidic channel.

In aspect 21, according to aspects 1 to 20, the magnetizable layer caninclude multiple pieces of elongated low magnetic permeability material,each piece of low magnetic permeability material being arranged within acorresponding elongated gap in the high magnetic permeability material.In aspect 22, according to aspect 21, the multiple elongated gaps can bearranged in parallel. In aspect 23, according to any one of aspects 21to 22, a thickness of at least one gap can extend completely through athickness of the high magnetic permeability material. In aspect 24,according to any one of aspects 21 to 23, for one or more of the piecesof low magnetic permeability material, a width of each of the one ormore pieces narrows along the central longitudinal axis associated withthe corresponding piece.

In accordance with another general aspect 25 of the disclosure, thereare provided microfluidic devices that include one or more magnets, eachmagnet being operable to emit a magnetic field; a magnetizable layerarranged on a surface of the one or more magnets, in which themagnetizable layer includes a high magnetic permeability material and alow magnetic permeability material adjacent to or at least partiallybordering the high magnetic permeability material, in which a thicknessof the high magnetic permeability material is greater than 10 μm and asaturation flux density of the high magnetic permeability material isgreater than 0.2 T, in which the magnetizable layer is configured toinduce a gradient in the magnetic field of at least one of the magnets;and

a microfluidic channel arranged on a surface of the magnetizable layer,in which a central longitudinal axis of the microfluidic channel isarranged at an angle to or laterally offset from a an interface betweenthe low magnetic permeability material and the high magneticpermeability material.

In aspect 26 according to aspect 25, the devices further include adeflection channel, and an output channel separate from the deflectionchannel, in which both the output channel and the deflection channel arefluidly coupled to an outlet of the microfluidic channel.

In accordance with another general aspect 27 of the disclosure, thereare provided methods of sorting a target analyte using the microfluidicdevices of any one of aspects 20 to 24, and 26, in which the methodsinclude flowing a fluid sample through the microfluidic channel, thefluid sample including the target analyte and one or more magneticparticles bound to the target analyte; exposing, during operation of themicrofluidic device, the fluid sample to the gradient in the magneticfield, in which the gradient in the magnetic field deflects the targetanalyte toward the channel away from an initial fluid flow trajectory ofthe fluid sample; and collecting the target analyte at an output of thedeflection channel.

The foregoing and other embodiments can each optionally include one ormore of the following features, alone or in combination. For example, inaspect 28, according to aspect 27, the one or more magnetic particlescan include or be selected from the group consisting ofsuperparamagnetic beads, diamagnetic beads, ferromagnetic beads, andcombinations thereof. In aspect 29, according to any one of aspects 27to 28, a ratio of a size of the target analyte to a number of magneticparticles bound to the target analyte can be greater than approximately10 μm, or greater than approximately 15 μm, or greater thanapproximately 20 μm, or greater than approximately 25 μm. In aspect 30,according to any one of aspects 27 to 29, one or more magnetic particlescan have diameters less than or equal to approximately 0.5 μm, or lessthan or equal to approximately 0.1 μm. In aspect 31, according to anyone of aspects 27 to 30, one or more magnetic particles have magneticmoments less than or equal to approximately 35 kA/m.

In aspect 32, according to any one of aspects 27 to 31, the methodsfurther include cycling the magnetic field on and off.

In accordance with another general aspect 33 of the present disclosure,there are provided methods of fabricating microfluidic devices, in whichthe methods include providing one or more magnets, each magnet beingoperable to emit a magnetic field; forming a magnetizable layer adjacentto the one or more magnets, in which the magnetizable layer isconfigured to induce a gradient in the magnetic field of at least one ofthe magnets, the gradient being at least 10³ T/m at a position that isat least 20 μm away from a surface of the magnetizable layer; andforming a microfluidic channel on a surface of the magnetizable layer.

The foregoing and other embodiments can each optionally include one ormore of the following features, alone or in combination. For example, inaspect 34, according to aspect 33, forming the magnetizable layerincludes providing a layer of high magnetic permeability material andforming one or more elongated gaps in the high magnetic permeabilitymaterial. In aspect 35, according to aspect 34, providing the layer ofhigh magnetic permeability material can include casting the layer ofhigh magnetic permeability material, molding the layer of high magneticpermeability material, thermoforming the layer of high magneticpermeability material, or depositing the layer of high magneticpermeability material using sputtering, thermal deposition, plasmadeposition, electro-plating or electron-beam deposition. In aspect 36,according to aspect 34, providing the layer of high magneticpermeability material can include placing magnetic tape on a surface ofthe one or more magnets. In aspect 37, according to any one of aspects34 to 36, forming the one or more elongated gaps can include machiningthe layer of high magnetic permeability material. In aspect 38,according to aspect 37, the method further includes machining through anentire thickness of the layer of high magnetic permeability material.

In aspect 39, according to any one of aspects 34 to 38, the methodsfurther include filling the one or more elongated gaps with a lowmagnetic permeability material, e.g., a non-magnetic material, e.g.,filling the one or more elongated gaps comprises using injection moldingof the low magnetic permeability material or hot embossing of the lowmagnetic permeability material.

In aspect 40, according to any one of aspects 34 to 39, the methodsinclude arranging a central longitudinal axis of the microfluidicchannel at an angle with respect to at least one of the elongated gaps.

In aspect 41, according to any one of aspects 34 to 40, the methodsfurther include placing a low magnetic permeability substrate, e.g., anon-magnetic substrate, on a surface of the one or more magnets, andforming the magnetizable layer on a surface of the low magneticpermeability substrate, e.g., the non-magnetic substrate.

In accordance with another general aspect 42 of the present disclosure,there are provided microfluidic devices that include one or moremagnets, each magnet being operable to emit a magnetic field; amagnetizable layer adjacent to the one or more magnets, the magnetizablelayer including a first high magnetic permeability portion and at leastone low magnetic permeability portion; and a microfluidic channeladjacent to the magnetizable layer, in which the microfluidic channelincludes a first analyte isolation region, and a second analyteisolation region fluidly coupled to the first analyte isolation region,in which the magnetizable layer is configured to induce a gradient inthe magnetic field of at least one of the magnets, in which the magneticfield gradient induced in the first analyte isolation region isdifferent from the magnetic field gradient induced in the second analyteisolation region, and in which the magnetic field gradients induced inat least one of the first analyte isolation region and the secondanalyte isolation region is at least 10³ T/m at a position that is atleast 20 μm away from a surface of the magnetizable layer.

In accordance with another general aspect 43 of the present disclosure,there are provided microfluidic cartridges that include a magnetizablelayer, the magnetizable layer comprising a high magnetic permeabilitymaterial and a low magnetic permeability material adjacent to or atleast partially bordering the high magnetic permeability material; amicrofluidic channel arranged on a surface of the magnetizable layer, inwhich a central longitudinal axis of the microfluidic channel isarranged at an angle to or laterally offset from an interface betweenthe high magnetic permeability material and the low magneticpermeability material; and a passivation layer arranged on the surfaceof the magnetizable layer and forming a bottom surface to themicrofluidic channel, in which a surface of the magnetizable layer isconfigured to be removably secured to a one or more magnets.

In aspect 44, according to aspect 43, the surface of the magnetizablelayer includes protruding structures configured to be removably lockedinto corresponding sections of the one or more magnets.

In aspect 45, according to any one of aspects 43 to 44, wherein acentral longitudinal axis of the microfluidic channel is arranged at anangle to or laterally offset from a an interface between the lowmagnetic permeability material and the high magnetic permeabilitymaterial.

In aspect 46, according to any one of aspects 43 to 45, the low magneticpermeability material is arranged within a gap in the high magneticpermeability material or between separate pieces of the high magneticpermeability material.

In aspect 47, according to any one of aspects 43 to 46, a first surfaceof the high magnetic permeability material and a first surface of thelow magnetic permeability material are in direct contact with thepassivation layer, and a thickness of the low magnetic permeabilitymaterial as measured from the passivation layer is greater than athickness of the high magnetic permeability material.

In accordance with another general aspect 48 of the present disclosure,there are provided microfluidic instruments that include one or moremagnets, each magnet being operable to emit a magnetic field; and amagnetizable layer arranged adjacent to the one or more magnets, whereinthe magnetizable layer is configured to induce a gradient in themagnetic field of at least one of the magnets, and wherein themagnetizable layer comprises a high magnetic permeability material, anda low magnetic permeability material adjacent to or at least partiallybordering the high magnetic permeability material, wherein a surface ofthe magnetizable layer is configured to be removably secured to amicrofluidic cartridge.

In aspect 49 according to aspect 48, the surface of the magnetizablelayer comprises protruding structures configured to be removably lockedinto corresponding sections of the microfluidic cartridge.

In aspect 50, according to any one of aspects 48 to 49, the low magneticpermeability material is arranged within a gap in the high magneticpermeability material or between separate pieces of the high magneticpermeability material.

In aspect 51, according to any one of aspects 48 to 50, a first surfaceof the high magnetic permeability material and a first surface of thelow magnetic permeability material form the surface of the magnetizablelayer, and wherein a thickness of the low magnetic permeability materialas measured from the surface of the magnetizable layer is greater than athickness of the high magnetic permeability material.

In accordance with another general aspect 52 of the present disclosure,there are provided methods of sorting a target analyte using themicrofluidic devices or the microfluidic cartridges of any one ofaspects 1 to 26 and 42-47. The methods include flowing a fluid samplethrough the microfluidic channel, the fluid sample comprising the targetanalyte and one or more magnetic particles bound to the target analyte;exposing, during operation of the microfluidic device or themicrofluidic cartridge, the fluid sample to the gradient in the magneticfield, wherein the gradient in the magnetic field deflects a trajectoryof the target analyte away from an initial fluid flow trajectory of thefluid sample.

In aspect 53 according to aspect 52, the gradient in the magnetic fieldexerts a force on the target analyte in a first direction toward themagnetizable layer. In aspect 54, according to aspect 53, the gradientin the magnetic field also exerts a force on the target analyte in asecond direction different from the first direction. In aspect 55,according to aspect 3, a width of the gap is equal to or greater thanapproximately 100 nm, e.g., equal to or greater than approximately 500nm, equal to or greater than approximately 1 μm, equal to or greaterthan approximately 10 μm, equal to or greater than approximately 50 μm,equal to or greater than approximately 75 μm, or equal to or greaterthan approximately 100 μm.

In aspect 56 according to any one of aspects 1 to 26 and 42-51, themicrofluidic channel is curved. In aspect 57, according to any one ofaspects 1 to 26, 42-51, and 56, a boundary between the high magneticpermeability material and the low magnetic permeability material iscurved. In aspect 58, according to any one of aspects 48-51, theinstrument is configured to be removably fixed to the microfluidiccartridge to any one of a plurality of different orientations withrespect to the microfluidic cartridge.

In aspect 59, according to any one of aspects 1 to 58, the high magneticpermeability material has a relative permeability that is greater thanthe relative permeability of the lower magnetic permeability material byat least about 4. In aspect 60, according to any one of aspects 1 to 59,the high magnetic permeability material has a relative permeability thatis at least about 5.

As used herein, “linked” means attached or bound by covalent bonds,noncovalent bonds, or other bonds, such as van der Waals forces.

As used herein, “specifically binds” means that one molecule, such as abinding moiety, e.g., an oligonucleotide or an antibody, bindspreferentially to another molecule, such as a target molecule, e.g., anucleic acid or protein, e.g., a cell surface marker, in the presence ofother molecules in a sample.

As used herein, “magnetic moment” is the tendency of a magnetic materialto align with a magnetic field.

As used herein, “saturation flux density” is the magnetic flux densityof a material when the material is fully magnetized, i.e., when there isa negligible increase in the flux density with further increases in amagnetizing field.

As used herein, “magnetizable” is understood to mean capable of beingmagnetized.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods, materials,and devices similar or equivalent to those described herein can be usedin the practice or testing of the present invention, suitable methods,materials and devices are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional view of an example of a microfluidic deviceas described herein.

FIG. 1B is a top view of a magnetizable layer of the microfluidic deviceof FIG. 1A.

FIG. 1C is a cross-sectional view of an example of a microfluidic devicethat includes a thin-film layer between a magnetizable layer and amicrofluidic cover.

FIG. 2 is a side view of an example of a microfluidic device asdescribed herein.

FIG. 3A is a cross-sectional view of a microfluidic device in which alow magnetic permeability region is laterally offset from a centrallongitudinal axis of the microfluidic channel.

FIG. 3B is a top view of the magnetizable layer of the microfluidicdevice of FIG. 3A.

FIG. 4 is a top view of a microfluidic device, with the cover removed,in which a low magnetic permeability region extends at an angle withrespect to a flow direction of a microfluidic channel.

FIG. 5A is a cross-sectional view of an example of a microfluidic devicehaving a magnetizable layer in which multiple low magnetic permeabilityregions are formed and arranged in parallel.

FIG. 5B is a top view of an example of a microfluidic device with acover removed and showing a surface of a magnetizable layer.

FIG. 5C is a schematic of the flow of labeled and unlabeled cells in amicrofluidic design that includes multiple parallel low magneticpermeability regions.

FIG. 5D is a schematic of the flow of labeled and unlabeled cells in amicrofluidic design that includes a single low magnetic permeabilityregion.

FIG. 6A is a cross-sectional view of an example of a microfluidicdevice, in which a microfluidic cover includes multiple microfluidicchannel regions.

FIG. 6B is a top view of the microfluidic device of FIG. 6A.

FIG. 7 is a cross-sectional view of a microfluidic device in which a lowmagnetic permeability region does not extend all the way to a bottomsurface of the magnetizable layer.

FIGS. 8A-8H are cross-sectional views of different arrangements ofmagnets around a magnetizable layer of a microfluidic device.

FIGS. 9A-9D are schematics of an example of a system that includes amicrofluidic device for isolating and/or sorting target analytes basedon high magnetic flux gradients.

FIGS. 10A-10D are schematics of an example of a system that includes amicrofluidic device as described herein for isolating and/or sortingtarget analytes based on high magnetic flux gradients.

FIG. 11 is a schematic of an example arrangement for a multistage devicethat utilizes high magnetic flux gradients to isolate target analytes.

FIG. 12A is a schematic of an example of a system that includes twoseparate stages for analyte isolation based on the use of magnetic fluxgradients.

FIG. 12B is a close-up view of the two separate stages for analyteisolation shown in FIG. 12A.

FIG. 13 is a flowchart describing a method for fabricating microfluidicdevices described herein.

FIG. 14A is a schematic of a first configuration of an example of amicrofluidic device that includes a removable and/or replaceableportion.

FIG. 14B is a schematic of a second configuration of an example of amicrofluidic device that includes a removable and/or replaceableportion.

FIGS. 15A and 15B are bar graphs that show the bead load and beadload-yield, respectively, for a stripe device fabricated according toTable 1.

FIG. 16 is a schematic cross-section of an integrated microfluidicdevice.

FIG. 17 is a heat map showing a top view of a simulated magnetic fieldgradient in a first isolation stage of an integrated microfluidicdevice.

FIG. 18 is a heat map showing a top view of a simulated magnetic fieldgradient in a second isolation stage of an integrated microfluidicdevice.

FIGS. 19A-19C are schematics showing cross-sections of three differentexample configurations of a magnetizable layer.

FIGS. 20A-20B are heat map plots of a simulated magnetic field gradientin a first and second isolation stage of an integrated microfluidicdevice.

FIGS. 21A-21C are schematics of different examples of configurations forthe arrangement of magnets in an integrated microfluidic device.

FIG. 22 is a schematic of a cross-section of a portion of an integratedmicrofluidic device excluding the fluidic channels and magnets.

FIGS. 23A-23B are each a schematic depicting a top view of a first andsecond stage of an integrated microfluidic device.

FIGS. 24A-24B are plots of the magnitude of a simulated magnetic fluxgradient in a first isolation stage and a second isolation stage,respectively, as a function of saturation flux density.

FIGS. 25A-25B are plots of the magnitude of a simulated magnetic fluxgradient in a first isolation stage and a second isolation stage,respectively, as a function of passivation layer thickness.

FIGS. 26A-26B are plots of the magnitude of a simulated magnetic fluxgradient in a first isolation stage and a second isolation stage,respectively, as a function of a thickness of a high magneticpermeability material (e.g., an alloy material).

FIGS. 27A-27F are schematics depicting top views of examples ofdifferent microfluidic channel designs and high magnetic flux gradientinducing structures.

FIGS. 28A-28F are schematics depicting top views of examples ofdifferent arrangements of a microfluidic channel with respect to a highmagnetic flux gradient inducing structure.

DETAILED DESCRIPTION

The present disclosure relates to methods and systems for sortingparticles using high gradient magnetic fields. In general, one aspect ofthe present disclosure can be embodied in microfluidic devices thatemploy high magnetic field gradients for sorting target agents oranalytes (e.g., nucleic acids, polypeptides, bacteria, cells) flowingwithin a microfluidic channel of the device. The devices can include oneor more magnets and a layer having one or more high magneticpermeability regions adjacent to or at least partially bordering a lowmagnetic permeability region. The high magnetic permeability regionprovides a preferred path for flux lines emanating from the one or moremagnets, such that the magnetic field lines extend over the low magneticpermeability region to establish a fringing flux field with a high fieldgradient. The high field gradient gives rise to a magnetic force that“pulls” the magnetic particles (and the analyte to which the magneticparticles are attached) flowing within the adjacent microfluidic channeltoward the gap for sorting.

FIG. 1A is a cross-sectional view of an example of a microfluidiccapable of generating high magnetic gradients for isolating targetanalytes. A Cartesian coordinate system is provided for reference, inwhich the positive z-direction is into the page. The device 100 includesone or more magnets 102, a magnetizable layer 104, and a microfluidicchannel cover 110 that defines a microfluidic channel region 112 throughwhich a sample fluid may flow. The device may optionally include aspacer layer 114 between the one or more magnets 102. The magnetizablelayer 104 may be composed of separate portions: a high magneticpermeability portion 106 having a magnetic permeability that is higherrelative to an elongated low magnetic permeability portion 108. The highmagnetic permeability portion preferably has a relative permeability(the ratio of the permeability of the medium to that of free spacepermeability) that is greater than the relative permeability of thelower magnetic permeability material by at least about 4. The highmagnetic permeability material preferably has a relative permeability ofat least 5. During operation of the device 100, a fluid sample may flowthrough the channel region 112, e.g., along the z-direction.

FIG. 1B is a top view of the magnetizable layer 104 with the cover 110removed. As shown in FIG. 1B, the elongated low magnetic permeabilityportion 108 also extends along the z-direction.

One of the principles behind the operation of device 100 is that themagnetizable layer 104 is configured to drive a large magnetic flux intoa small region of space, thus giving rise to a high flux density, andtherefore a strong magnetic force within the channel region 112. Inparticular, the high magnetic permeability portion 106 of layer 104provides the preferred (low reluctance) path for the magnetic fluxemanating from the one or more magnets 102. In contrast, the magneticflux tends to avoid the low magnetic permeability portion 108 locatedbetween the high permeability regions 106. In addition, the region abovethe magnetizable layer 104 (e.g., the microfluidic cover 110 includingthe channel region 112) preferably also has a low magnetic permeabilityrelative to the portion 106. As a result of the tendency of the magneticflux to prefer the high magnetic permeability regions, a portion of theflux passes through the surface of the high permeability portion 106 ona first side of the low permeability region 108, through themicrofluidic channel 112, and then back into the high permeabilityportion 106 on a second opposite side of the low permeability region108. The portion of the flux extending into the microfluidic channel 112is the “fringing” or “leakage” flux (field).

The magnitude of the fringing flux that extends into the channel 112 isvery large close to the surface of the magnetizable layer 104, but dropsoff quickly as the distance from the magnetizable layer surfaceincreases, giving rise to a large flux gradient. The force on a magneticparticle passing through the channel is proportional to the magnitude of∇B, where ∇ corresponds to the vector differential operator and B is themagnetic flux. The gradient in flux may be largest at the edges betweenthe high magnetic permeability portions 106 and the low magneticpermeability portions 108. Therefore, magnetic particles (and analytesattached to the magnetic particles) that flow through the channel 112 inthe region above the low magnetic permeability portion 108 are attractedby the strong magnetic force towards the surface of the magnetizablelayer 104. In contrast to a device in which a homogeneous magnetic fieldextends throughout the fluidic channel, the magnetic field gradientenables, in certain implementations, a greater “pull” on magneticparticles, thus allowing quicker isolation of magnetically labeledanalytes from other analytes in the fluid sample. As a result, highersample fluid velocities and/or shorter fluidic channels lengths can beused for separating the target analytes.

FIG. 2 is a side view of an example of the device 100 taken at line A-Aof FIG. 1. FIG. 2 depicts how magnetic particles can be sorted from afluid sample 200 using the high magnetic field gradient. A coordinatesystem is again shown for reference. The fluid sample 200 enters thedevice 100 from the left (as indicated by the arrow). The sample 200contains both magnetic particles 202 and non-magnetic particles 204 in afluid such as water, a buffer, saline, blood (e.g., diluted blood orwhole blood), or other applicable fluid. For the present example, thefluid sample 200 follows a pressure-driven laminar flow having aparabolic velocity profile, with the highest fluid velocity at thecenter of the channel 112 and low fluid velocity near the boundary wallsof the channel 112. However, other fluid driving mechanisms, such aselectrokinetic techniques, having different velocity profiles can alsobe used.

As the fluid sample 200 passes over the magnetizable region 104, thehigh magnetic gradient generated by the device gives rise to a magneticforce that pulls magnetic particles 204 from the sample 200 toward thesurface of the region 104. Non-magnetic particles 202 are unaffected bythe magnetic force and continue flowing with the sample 200 atapproximately the same height in the channel 112. The separation of themagnetic particles 204 from the non-magnetic particles 202 enablesindependent collection of the magnetic particles and/or analytesattached to the magnetic particles 204.

Referring again to FIG. 1A, the greater the magnetic flux driven throughthe high permeability region 106, the stronger the magnetic force in theregion above the low magnetic permeability region 108 and, therefore,the stronger the force experienced by magnetic particles traveling inthe vicinity of the low magnetic permeability region 108. The magnitudeof flux can be affected based on one or more parameters of themicrofluidic device 100 including, for example, the strength of themagnet(s) that emits the magnetic field. The stronger the magnet used,the greater the flux that can be achieved. The strength of the maximummagnetic field that can be produced from a magnet is denoted using thesymbol Br, i.e., the remanent magnetization of the magnet. The types ofmagnets that may be used include, for example, permanent magnets orelectromagnets. The magnets may be composed of material including, forexample, alloys of NdFeB, SmCo, AlNiCo, or ferrite. The magnetic fieldprovided by the one or more magnets 102 may be in the range ofapproximately 0.001 T to approximately 1.5 T. For example, the magneticfield emitted by the one or more magnets 102 may be approximately 0.1 T,approximately 0.3 T, approximately 0.5 T, approximately 1 T, orapproximately 1.3 T. Other values for the magnetic field are possible aswell.

Another parameter that affects the flux magnitude, and therefore theflux gradient that can be achieved, is the maximum magnetic permeabilityof region 106, i.e., the saturation flux density Bs. The greater thesaturation flux density, the greater the amount of flux that can passthrough the region 106 leading to gains in flux gradient. Accordingly,it is preferable that the amount of flux through the high relativemagnetic permeability region 106 is at least equal to or greater thanthe saturation flux density Bs of the material forming region 106.Materials that can be used for the high magnetic permeability region 106include, but are not limited to, iron, nickel, cobalt, and nickel-ironalloys such as Ni₈₀Fe₂₀ or Ni₄₅Fe₅₅, steel, CoFeNi, FeAlN alloys, SiFealloys, or CoFe alloys. The high magnetic permeability materials mayhave saturation permeabilities that are greater than or equal toapproximately 1 T. For example, the high magnetic permeability materialmay have a saturation flux density that is greater than or equal toapproximately 1.2 T, greater than or equal to approximately 1.4 T,greater than or equal to approximately 1.6 T, greater than or equal toapproximately 1.8 T, or greater than or equal to approximately 2.0 T.

The parameters Br and Bs may also be used to determine other propertiesof the microfluidic device 100 that enable achieving high flux gradientsabove the low magnetic permeability region 108. For example, to a firstorder approximation, the relationship between remnant magnetization andsaturation flux density of the high magnetic permeability region shouldfollow (Br)w_(m)≧(Bs)(hs), where w_(m) is the cross-sectional width ofthe one or more magnets 102 and hs is the height of the magnetizablelayer 104. Thus, the minimum cross-sectional width that should be usedto obtain a maximum flux gradient for a particular magnet/highpermeability material combination may be expressed as w_(m)≧(Bs/Br)hs.Examples of cross-sectional width w_(m) can range from approximately 1μm to approximately 50 mm, including, for example, approximately 50 μm,approximately 500 μm, approximately 1 mm, approximately 2 mm,approximately 5 mm, or approximately 10 mm. Similarly, a magnetthickness hm approximately equal to the cross-sectional width should bemore than sufficient to obtain the maximum flux gradient. Examples ofmagnet thicknesses are approximately 500 μm, approximately 1 mm,approximately 2 mm, approximately 4 mm, approximately 5 mm, orapproximately 10 mm.

A thickness hs of the magnetizable layer 104 may fall within the rangeof approximately 1 μm to approximately 10 mm. For example, the thicknesshs may be approximately 10 μm, approximately 100 μm, approximately 250μm, approximately 500 μm, approximately 1 mm, or approximately 5 mm.Other thicknesses are possible as well. The thickness of the highmagnetic permeability material 106 may be equal to or less than athickness of the magnetizable layer 104. For example, a thickness of thehigh magnetic permeability material 106 may be approximately 1 μm, 10μm, approximately 100 μm, approximately 250 μm, approximately 500 μm,approximately 1 mm, approximately 5 mm, or approximately 10 mm. Otherthicknesses for the high magnetic permeability region are possible aswell. Preferably, the width ws of the magnetizable layer 104 issufficient to accommodate the width of the one or more magnets 102beneath layer 104, as well as the width of the channel 112 above thelayer 104, and may fall within the range of approximately 500 μm toapproximately 100 mm. For example, the width ws may be approximately 1mm, approximately 5 mm, approximately 10 mm, approximately 25 mm,approximately 50 mm, or approximately 75 mm. Other widths are possibleas well. In some implementations, the width ws may extend beyond 100 mmto accommodate multiplexing in which multiple channels 112 are formedwithin the device 100, e.g., in parallel or in series.

An appropriate length ls for the magnetizable layer 104 (and, in somecases, the length of the channel region 112 and magnet(s) 102) forisolating target analytes depends on various factors including, amongother things, the strength of the magnetic force in the channel region112, the residence time of analytes within the channel 112, the velocityor flow rate of the fluid sample as it passes through the channel 112,and the responsivity of magnetic particles to the magnetic force in thechannel 112. The length ls of the magnetizable layer may fall within therange of approximately 1 mm to approximately 500 mm. For example, thelength is may be approximately 5 mm, approximately 10 mm, approximately50 mm, approximately 100 mm, approximately 250 mm, approximately 500 mm,or approximately 750 mm. Other lengths are possible as well. The lengthof the high magnetic permeability material 106 may be equal to or lessthan a length of the magnetizable layer 104. For example, a length ofthe high magnetic permeability material 106 may be approximately 1 mm, 5mm, approximately 10 mm, approximately 50 mm, approximately 100 mm,approximately 250 mm, approximately 500 mm, or approximately 750 mm.Other lengths for the high magnetic permeability region are possible aswell.

Though the high magnetic permeability region 108 is shown as having arectangular cross-section in FIG. 1A, other shapes are also possible forregion 108. For example, the region can have any number of differentcross-sections, e.g., square, circular, triangular, hexagonal, curved,e.g., concave or convex, or oval cross-sections. Other cross-sectionsare possible. The important aspect is to create an interface between thehigh magnetic permeability material and the low magnetic permeabilitymaterial.

Preferably, the low magnetic permeability region 108 has a substantiallysmaller relative magnetic permeability than that of the high magneticpermeability region 106. For example, the low magnetic permeabilitymaterial has a relative magnetic permeability that is lower than therelative magnetic permeability of the high magnetic permeabilitymaterial by at least about 4. Various materials can be used as the lowmagnetic permeability region 108. For example, the low magneticpermeability portion 108 can include, but is not limited to, polymers(e.g., polyethylene, polyimide, polymethamethacrylate (PMMA),polystyrene, polydimethylsiloxane (PDMS), epoxy), glass, ceramics, metal(e.g., brass), or silicon. The low magnetic permeability material caninclude non-magnetic materials. The materials to be included in the lowmagnetic permeability portion 108 are not limited to solid materials andinclude fluids such as water. In some implementations, portion 108 maycorrespond to a gap comprising air, a gas (e.g., an inert gas), or avacuum. The relative magnetic permeability of the portion 108 may rangefrom approximately 1 to approximately 1000. As explained above, thesaturation flux of the portion 108 should be less than the saturationflux of the portion 106. Preferably, the difference in saturation fluxis about 1 T, though the difference can be greater or less than 1 T.

Preferably, the flux gradient is widely distributed across the width ofthe channel 112 so that even magnetic particles at a relatively fardistance from the surface of the magnetizable layer 104 (e.g., at thetop of the channel 112) experience the magnetic force and are “pulled”down toward the layer 104, and more specifically, toward the elongatedlow magnetic permeability portion 108 where the flux gradient ishighest. Preferably, the width of the low magnetic permeability portion108 is approximately the same size as or larger than the width of thetarget analyte. For example, for a typical cell of approximately 20 μm,the width of the portion 108 is also approximately 20 μm. The width ofthe portion 108 may fall within the range of approximately 100 nm toapproximately 500 μm. For example, the width of the portion 108 may beapproximately 500 nm, approximately 1 μm, approximately 10 μm,approximately 50 μm, or approximately 75 μm. Similarly, the width of thechannel region 112 is preferably just as wide or wider than the width ofthe low magnetic permeability portion 108 to allow desired targetanalytes to pass through. In addition, the width of the channel region112 may be wider or narrower than the width of the elongated portion108.

In some implementations, the width of the low magnetic permeabilityportion 108 may vary across the length is of the magnetizable layer 104.For example, in some cases, the width of the portion 108 may be taperedfrom a first side of the magnetizable layer 104 toward a second oppositeside of the magnetizable layer 104 along the z-direction. Alternatively,or in addition, the width of the low magnetic permeability portion 108may vary across the thickness hs of the magnetizable layer 104. Forexample, in some cases, the width of the portion 108 may be tapered froma top surface of the magnetizable layer 104 near the channel region 112toward a bottom surface of the magnetizable layer 104 near the optionalspacer layer 114 or vice versa. Tapering the width of portion 108 fromthe base of the magnetizable layer 104 to the top of the magnetizablelayer near the channel region 112 may, in some implementations, providethe advantage of a more spatially distributed flux gradient in thechannel 112.

In some implementations, the magnetizable layer 104 may be composed offlexible magnetic tape in the form of a strip or foil, in which the tapematerial is used as the high magnetic permeability material. Flexiblemagnetic tape offers the advantage, in some cases, of being easy toapply to the one or more magnets 102 or the spacer layer 114. Anadhesive may be formed on one side of the magnetic tape for adhering tothe one or more magnets 102 or the spacer layer 114. Examples ofmaterial that may be used for flexible magnetic tape include, but arenot limited to, Master Magnetics magnetic tape (Master Magnetics, Inc.)or Magna Card magnetic tape (Magna Card, Inc.). The low magneticpermeability portion 108 may be formed in the magnetic tape by etchingor cutting out a pre-defined region of the magnetic tape.

The microfluidic cover 110 can be formed from any applicable materialthat is compatible with the fluid sample to be delivered through themicrofluidic channel. For example, the microfluidic cover 110 can beformed of glass, silicon, PDMS, PMMA, cyclo olefin polymer (COP),polycarbonate, polyimide, or other suitable material. The spacer layer114 is optional and may serve as a supporting layer on which themagnetizable layer 104 may be formed. The spacer layer 114 may be formedof low magnetic permeability material, e.g., a non-magnetic materialincluding, for example, glass or any of a wide variety of plastics.

In some implementations, an additional layer of material may be formedon the surface of the magnetizable layer 104. For example, a layer ofmaterial may be formed on the surface of the magnetizable layer 104 toprotect the magnetizable layer 104 from damage and/or corrosion from thefluid sample, to provide a surface on which target analytes may bebound, to provide a surface to which the microfluidic cover 110 can beadhered, and/or to isolate the low-permeability region 108 from thefluid sample. FIG. 1C is a cross-sectional view of an examplemicrofluidic device 150 that includes a thin-film layer 152 between themagnetizable layer 104 and the microfluidic cover 110. The thin-filmlayer 152 may be formed of one or more various materials including, butnot limited to, a silicon material, such as silicon dioxide, glass, orany of a wide variety of inert plastics. The thin-film layer 152 mayhave a thickness in the range of approximately several nanometers toseveral tens of microns. For example, the thin-film layer 152 may beapproximately 10 nm thick, approximately 100 nm thick, approximately 500nm thick, approximately 1 μm thick, or approximately 5 μm thick. Otherthicknesses may be used as well.

In the example shown in FIG. 1A, the low magnetic permeability portion108 is substantially aligned with a central longitudinal axis of themicrofluidic channel 112. The central longitudinal axis of the channel112 extends along the z-axis into the page and is equidistant from thewalls of the channel 112. Similarly, the central longitudinal axis ofthe elongated low magnetic permeability portion 108 also extends alongthe z-direction. Other configurations of the channel 112 and lowmagnetic permeability portion 108 are also possible and, in someimplementations, may enhance the isolation of magnetic or magneticallylabeled particles from a fluid sample.

For example, in some implementations, the low magnetic permeabilityregion 108 may be laterally offset from the central longitudinaldirection of the microfluidic channel 112. FIG. 3A is a cross-sectionalview of a microfluidic device 300 in which a low magnetic permeabilityregion 108, bounded by the high magnetic permeability regions 106, islaterally offset from a central longitudinal axis of the microfluidicchannel 112. FIG. 3B is a top view of the magnetizable layer 104 of themicrofluidic device 300 with the cover 110 removed. The dashed line 113in FIG. 3B represents the central longitudinal axis of the channel 112.As shown in these examples, the low magnetic permeability region 108 islocated closer to a sidewall 115 of the channel 112 than to a center ofthe channel, in which the channel center corresponds to the centrallongitudinal axis 113. The flux gradient established by the regions 106and 108 still gives rise to a magnetic force that pulls magneticparticles flowing through the channel downward toward the surface of themagnetizable layer 104. Because of the lateral offset between theportion 108 and the channel 112, however, magnetic particles flowingthrough the fluidic channel 112 will also be pulled laterally in thedirection of the low magnetic permeability region 108. Given asufficient distance along the flow direction of the channel 112, themagnetic particles can be substantially isolated from other particlesand/or analytes in the sample. In some cases, the channel 112 mayinclude a bifurcation at one end, such that the substantially isolatedparticles follow one path of the bifurcation whereas the remainingportion of the sample follows the other path of the bifurcation.

In some implementations, the elongated low magnetic permeability regioncan be arranged at an oblique angle with respect to the centrallongitudinal axis of the microfluidic channel. FIG. 4 is a top view of amicrofluidic device 400, with the cover 110 removed, in which a lowmagnetic permeability region 108, bounded by the high magneticpermeability regions 106, extends at an angle θ with respect to a flowdirection of the microfluidic channel 112, or with respect to a centrallongitudinal axis 113 of the microfluidic channel 112, e.g., at anoblique or acute angle. Again, the regions 106 and 108 induce a fluxgradient that acts as a magnetic force to pull magnetic particles towardthe portion 108 and away from other analytes flowing within the sample.The angle θ can be anywhere between, e.g., 0° to 30°, including, forexample, about 0.25°, about 0.5°, about 1.5°, about 5°, or about 15°.

The magnetizable layer 104 is not limited to having a single elongatedlow magnetic permeability region. Instead, in some implementations, themagnetizable layer 104 can include multiple elongated low magneticpermeability regions 108 arranged in parallel, each region 108 beingseparated from an adjacent region 108 by the high magnetic permeabilityportion 106. In this way, the width of the flux gradient can besubstantially extended. As a result, wider microfluidic channels 112 canbe used in which the flux gradient extends across the width of thechannels.

FIG. 5A is a cross-sectional view of an example of a microfluidic device500 having a magnetizable layer 104 in which multiple low magneticpermeability regions 108 are formed and arranged in parallel. As inother examples, a microfluidic cover 110 is formed over the magnetizablelayer 104 and includes a microfluidic channel 112. The distance betweenthe portions 108 (i.e., the width of the high magnetic permeabilityportion 106 between portions 108) may be similar to the widths ofportions 108 and in the range of approximately 100 nm to approximately100 μm. For example, the width of the portions 106 located between lowmagnetic permeability regions 108 may be approximately 500 nm,approximately 1 μm, approximately 10 μm, approximately 50 μm, orapproximately 75 μm. Other widths are possible as well.

In some implementations, the multiple low magnetic permeability portions108 may be arranged in parallel as well as at an oblique angle withrespect to a central longitudinal axis of the microfluidic channel 112.FIG. 5B is a top view of an example of a microfluidic device 550 withthe cover 110 removed and showing a surface of the magnetizable layer104. In this implementation, the magnetizable layer 104 includesmultiple elongated low magnetic permeability portions 108 arranged inparallel and at an oblique angle with respect to a central longitudinalaxis 113 of the microfluidic channel. As with the single low magneticpermeability region design, the parallel set of low magneticpermeability regions 108 induce a flux gradient that acts as a magneticforce to pull magnetic particles toward the magnetizable layer 104 andaway from a central longitudinal axis of the microfluidic channel 112.

FIGS. 5C-5D are schematics depicting the directional flow of labeled andunlabeled cells in a microfluidic design that includes multiple parallellow magnetic permeability regions (“stripes”) and in a microfluidicdesign that includes a single low magnetic permeability region (“gap”).The schematics show top views of the deflection channel region. Labeledcells move along the magnetizable structures, at a slight angle, e.g.,at about 0.25°, about 0.5°, about 1.5°, about 10°, or about 15° relativeto the direction of fluid flow, while unlabeled cells move in thedirection of fluid flow in the channel.

Alternatively, or in addition, multiple microfluidic channels 112 can beformed on the magnetizable layer 104. Multiple channel regions 112 allowa greater number of fluid samples and/or a greater amount of fluidsample to be examined simultaneously, thus enhancing the efficiency ofthe microfluidic device. In some implementations, the multiple channelregions 112 may be arranged in parallel such that they each cross over aportion of the one or more low magnetic permeability regions of themagnetizable layer 104.

FIG. 6A is a cross-sectional view of an example of a microfluidic device600, in which the microfluidic cover 110 includes multiple microfluidicchannel regions 112 a, 112 b, 112 c. FIG. 6B is a top view of themicrofluidic device 600. Though the channels 112 a, 112 b, 112 c are notshown in FIG. 6B, the central longitudinal axis 113 a, 113 b, 113 c ofeach channel 112 a, 112 b, 112 c is indicated by means of a dashed line.As shown in these examples, a single elongated low magnetic permeabilityregion 108 is used in the magnetizable layer. However, the low magneticpermeability region 108 is arranged at an oblique angle with respect toa central longitudinal axis of each of the microfluidic channels, suchthat the low magnetic permeability region 108 crosses over each of thechannels 112 as one moves from a first side of the microfluidic deviceto a second side of the microfluidic device. Though a single lowmagnetic permeability region 108 is shown in FIGS. 6A and 6B, multiplelow magnetic permeability regions can be used in the magnetizable layer104, for example in parallel.

As shown in FIG. 1A, the low magnetic permeability region 108 has athickness equal to a thickness of the magnetizable layer 104. That is,the low magnetic permeability region 108 extends from a top surface ofthe magnetizable layer 104 to a bottom surface of the layer 104. In someimplementations, the low magnetic permeability region 108 may extendonly through a portion of the magnetizable layer 104 that is thinnerthan the thickness of layer 104. For example, FIG. 7 is across-sectional view of a microfluidic device 700 in which the lowmagnetic permeability region 108 does not extend all the way to a bottomsurface of the magnetizable layer. Instead, the portion 108 is exposedon its bottom surface to the high magnetic permeability material 106. Inthe example device shown in FIG. 7, it is preferable that the highmagnetic permeability region 106 a beneath the low magnetic permeabilityregion 108 is saturated with the magnetic flux from the magnets 102.This is because when a high magnetic permeability material saturates,the relative permeability of the material approaches one. Thus, bysaturating the region 106 a beneath the portion 108, the portion 108 andregion 106 a behave similar to a low magnetic permeability portion 108that extends completely from a top surface of the magnetizable layer 104to a bottom surface of the magnetizable layer 104. Failure to saturatethe region 106 a may substantially degrade the flux gradient that can beachieved in the channel region 112.

Different configurations of the magnetizable layer can also affect thefield gradients that result in the fluidic channels. For example, FIGS.19A to 19C are a series of schematics that show cross-sections of threedifferent possible configurations of the magnetizable layer. FIG. 19Ashows a cross-section of the fluidic device, in which the magnetizablelayer 1904 is comprised mainly from high magnetic permeability material1906, with the exception of the gap region containing the low magneticpermeability material 1908.

FIG. 19B shows a cross-section of the fluidic device, in which the gapis still present, but the portions of the magnetizable layer 1904containing the high magnetic permeability material 1906 have beenreduced in size, relative to the fluidic channels, such that the lowmagnetic permeability material 1908 surrounds the high magneticpermeability material 1906 in the magnetizable layer 1904. Furthermore,the separation between the microfluidic channels has been increased sothat the first isolation stage (“S1”) is no longer positioned directlyover the high magnetic permeability material 1906. In addition, thefluidic channel cover 1910 that defines the first isolation stage andthe second isolation stage (“S2”) can be formed of the same low magneticpermeability material as the gap material (or a material having similarlow magnetic permeability). For example, the microfluidic cover and thelow magnetic material both can be formed from plastic, e.g., from thesame type of plastic.

FIG. 19C shows a cross-section of a fluidic device similar to that shownin FIG. 19B, except that there is only one region containing a highmagnetic permeability material 1906, thus widening the “gap” to the fullextent of the device. In other words, this is a one-sided “gap” that isreferred to herein as the “edge” configuration. For example, the highmagnetic permeability region 1906 may be a single piece of a magneticalloy with an edge 1907. Though not shown, the low and high magneticpermeability materials in each of FIGS. 19A-19C are assumed to extendinto and out of the page.

Referring back to FIG. 1A, this figure shows an array of two magnets 102arranged such that each magnet 102 has a magnetic pole orientation thatis opposite to a magnetic pole orientation of the adjacent magnet 102.In this arrangement, the magnetic field of each permanent magnet in thearray extends to an adjacent permanent magnet in the array. The numberof magnets used in the array is not limited to two, however, and may beincreased based on the design requirements of the microfluidic device.Alternatively, a single magnet can be used to provide the magnetic fluxnecessary for creating the flux gradient. In some implementations, themagnets can be arranged so that the magnetic poles of adjacent magnetsare in the same orientation, instead of an opposite orientations. Insome implementations, the magnets are arranged above or on the sides ofthe magnetizable layer 104.

FIGS. 8A-8H are cross-sectional views of different arrangements ofmagnets 102 around a magnetizable layer 104 of a microfluidic device, inwhich each magnet 102 has a “north” magnetic pole, labeled “N,” and a“south” magnetic pole, labeled “S.” Each of the implementations shown inFIGS. 8A-8H is capable of generating a magnetic flux that can be used bythe magnetizable layer to create a flux gradient in the channel region112 of a microfluidic device.

When increasing the distance separating the microfluidic channels in thefirst and second stages, the gradient in the magnetic flux should be bigenough in the channel positioned far from the gap or edge to stillinduce some deflection of magnetic particles in that channel. To ensurea sufficient flux, additional magnets can be placed in the vicinity ofthe channel that is positioned relatively far from the gap or edge. Forexample, for the device configuration shown in FIG. 21A (similar to theconfiguration shown in FIG. 19A), a sufficient flux gradient can beprovided with just two magnets 2150 beneath the gap region. In contrast,for the gap configuration shown in FIG. 21B (similar to theconfiguration shown in FIG. 19B), an additional magnet 2152 can be usedto generate magnetic flux in the region corresponding to the firstisolation stage S1. In some implementations, further magnets can beadded, for example, above the fluidic device in addition to below thedevice, as shown in FIG. 21B. Similar magnet arrangements would besuitable for the edge and other configurations, such as shown in FIG.21C.

The different arrangements of the microfluidic device described hereinare capable of producing extremely high flux gradients at distancesrelatively far from the surface of the magnetizable layer that isclosest to the microfluidic channel. For example, in someimplementations, the magnetizable layer 104 is capable of creating aflux gradient that is at least 10³ T/m at a position that is at least10, 20, 30, 40, or at least 50 μm away from a surface of themagnetizable layer 104. In some implementations, the magnetizable layer104 is capable of creating a flux gradient that is at least 10⁴ T/m at aposition that is at least 20, 30, 40, or at least 50 μm away from asurface of the magnetizable layer 104.

Depending on the thickness of any interlayers between the bottom surfaceof the microfluidic channel and the top surface of the magnetizablelayer, the device can also product high flux gradients at distancesrelatively far from the bottom surface of the microfluidic channel. Forexample, in some implementations, the magnetizable layer 104 is capableof creating a flux gradient that is at least 10³ T/m at a position thatis at least 10, 20, 30, 40, or at least 50 μm away from a bottommicrofluidic channel surface. In some implementations, the magnetizablelayer 104 is capable of creating a flux gradient that is at least 10⁴T/m at a position that is at least 20, 30, 40, or at least 50 μm awayfrom a surface of the a bottom microfluidic channel surface.

The high flux gradients that are obtainable with the devices describedherein have several advantages. For example, in some implementations,the high flux gradients enable the isolation of target analytes bound tomagnetic particles having very low magnetic moments: the high magneticforce in the microfluidic channel exerts a greater “pull” on themagnetic particles having low magnetic moments. Alternatively, or inaddition, the high flux gradients enable the isolation of targetanalytes bound to a lower number of magnetic particles, because themagnetic force is high, fewer magnetic particles having a particularmagnetic moment are required to be bound to a target analyte.

In some implementations, the high flux gradient enables magneticallylabeled analytes to be separated/isolated at high flow rates (e.g., atleast approximately 50 μL/min, at least approximately 100 μL/min, atleast approximately 150 μL/min, at least approximately 300 μL/min, atleast approximately 500 μL/min, or at least approximately 1000 μL/min),thus increasing the efficiency with which the device can be used fordetection and separation of target analytes.

In some implementations, the high flux gradients enable the use ofshorter microfluidic channels/isolation regions (e.g., less thanapproximately 150 mm, less than approximately 100 mm, less thanapproximately 50 mm, less than approximately 10 mm, or aboutapproximately 1 mm) since magnetically labeled analytes can be separatedover much shorter distances.

In some implementations, the high flux gradients enable the low magneticpermeability region to be arranged at shallower angles with respect to acentral longitudinal axis of the microfluidic channel. By arranging thelow magnetic permeability region at shallower angles, the fluid can flowfaster through the microfluidic channel, while still achievingseparation of desired particles from undesired particles in the samplefluid. An advantage of faster flow is that clogging in the microfluidicchannel can, in certain implementations, be reduced. However, withshallower angles (and increased speed), the length of the microfluidicchannel must increase to achieve a specified lateral displacement ofdesired particles from undesired particles in the sample fluid. This isbecause the lateral speed of the particles being separated remainsessentially constant regardless of the angle of the low magneticpermeability region. Accordingly, the time required to achieve a givenlateral separation also remains constant.

The sample fluid flow rate v is approximately inversely proportion tosin(θ), where θ is the angle of the low magnetic permeability regionwith respect to a central longitudinal axis of the microfluidic channel.

One way to characterize a microfluidic device that isolates targetanalytes through the use of magnetic force is to specify a ratio,R_(a/p), of a size, A, of the target analyte to a minimum number ofmagnetic particles, P, bound to the target analyte that would berequired to isolate the target analyte. For the purposes of calculatingthe ratio, size is understood to correspond to an average diameter oraverage length of the target analyte. Implementations of themicrofluidic devices described herein can obtain, for example, analytesize to particle number ratios greater than or equal to approximately 1μm, greater than or equal to approximately 5 μm, greater than or equalto approximately 10 μm, greater than or equal to approximately 50 μm, orgreater than or equal to 100 μm.

FIG. 9A is a schematic of an example of a system 900 that includes amicrofluidic device for isolating and/or sorting target analytes basedon high magnetic flux gradients. The system includes a microfluidicchannel that extends from an inlet 902 through an inertial focusingstage 904 and a deflection channel 906 to an outlet 908. The inertialfocusing stage 904 focuses cells in the center of microfluidic channel.Examples and further discussion of inertial focusing can be found, forexample, U.S. Pat. No. 8,186,913, incorporated herein by reference inits entirety. A magnetizable layer is located beneath the deflectionchannel 906 and includes multiple elongated low magnetic permeabilityportions 108 arranged in parallel and embedded in a high magneticpermeability material. The angle of the low magnetic permeabilityregions 108 relative to the flow path of the microfluidic channel isabout 0° to about 30°, e.g., about 0.25, about 0.5, about 1, about 1.5,about 10, about 15 or about 25°. The outlet 908 splits into a wastechannel 910 and a target channel 912. One or more magnets (not shown)are placed adjacent to the system 100 and provide a magnetic field.

The three insets shown in FIGS. 9B, 9C, and 9D are close-up views of theregions 108 at the beginning, middle and end, respectively, of thedeflection channel 906. When a fluid sample containing target analytesbound to one or more magnetic particles is introduced into thedeflection channel 906, the magnetic force created by the magnetizablelayer pulls the magnetic particles (and the attached analytes) in adirection of the low magnetic permeability regions 108. The magneticparticles will accumulate near and follow the regions 108 over thelength of channel 906 such that when the fluid sample reaches the outlet908, the magnetic particles (and the attached analytes) are isolatedfrom other non-labeled analytes in the sample. The portion of the fluidsample containing the non-labeled analytes flows out into the wastechannel 910 whereas the magnetic particles flow into the target channel912. In the example shown in FIG. 9, the regions 108 are presumed to beapproximately 40 μm wide with a spacing of approximately 40 μm.

FIG. 10A is a schematic of an example of another system 1000 thatincludes a microfluidic device for isolating and/or sorting targetanalytes based on high magnetic flux gradients. The system 1000 issimilar to system 900 except that instead of multiple elongated lowmagnetic permeability regions in a magnetizable layer, the magnetizablelayer includes a single low magnetic permeability region 108. In theexample of FIG. 10A, the deflection channel 1006 angles up atapproximately 0.5° relative to the low magnetic permeability region 108such that the distance between the region 108 and the wall of thedeflection channel 1006 increases (or decreases) along the length of thedeflection channel 1006. The three insets shown in FIGS. 10B, 10C, and10D are close-up views of the regions 108 at the beginning, middle andend, respectively, of the deflection channel 1006.

The position of the gap (which provides two interfaces) or interface (asingle interface) between the low magnetic permeability material and thehigh magnetic permeability material does not have to be in the center ofthe microfluidic channel width. Instead, in some implementations, thegap (i.e., the low magnetic permeability region between the highmagnetic permeability materials) or the interface (i.e., the interfacebetween a high magnetic permeability material and a low magneticpermeability material) may have different arrangements. For example,FIGS. 27A to 27C are schematics depicting top views of examples ofdifferent fluidic channels and the structure used to induce the highgradient. Each figure shows an outline of a fluidic channel 2700 and alow magnetic permeability region 2702 such as is formed in the “gap”structure of FIG. 1A. Alternatively, the region 2702 could correspond tothe interface between a high magnetic permeability material and the lowmagnetic permeability material in the “edge” configurations of themicrofluidic device. As shown in FIG. 27A, the location of the gap oredge interface may be laterally offset from the walls of the channel2700, such that the gap or edge appears to be outside of the channelfrom a top view. Alternatively, the location of the gap or edge may belocated directly underneath the channel 2700 (e.g., see FIG. 27B).Alternatively, the gap or edge can be positioned at an oblique anglewith respect to a central longitudinal axis of the channel 2700 (e.g.,see FIG. 27C).

In some implementations, the channel 2700 itself can include somecurvature. The gap or edge of the magnetizable layer that induces thehigh gradient in the magnetic field can also be curved to substantiallyfollow the curvature of the channel 2700. For example, FIG. 27D is aschematic of a top view of a curved channel 2700, in which the gap/edgeregion 2702 of the magnetizable layer is also curved, but laterallyoffset from the channel 2700. FIG. 27E is a schematic of a top view of acurved channel 2700 in which the gap/edge region 2702 follows thecurvature of the channel 2700 and is also located directly beneath thechannel 2700. FIG. 27F is a schematic of a top view of a curved channel2700, in which the gap/edge region 2702 is curved and located beneaththe channel 2700, but does not follow a central axis of the fluidicchannel. Instead, as shown in FIG. 27F, the curved gap/edge region 2702is oriented obliquely with respect to the central axis of the fluidicchannel 2700.

Potential risks of using magnetizable structures that generate largeflux gradients within microchannels is clogging with the magneticparticles or with heavily magnetic particle-laden analytes, which arevery strongly attracted to the magnetizable layer. Negative effects ofthis clogging include a reduction in the quality of a focused stream ofanalytes and/or loss of the target analytes (e.g., through lysis oftarget cells). The risks of clogging can be mitigated through the use ofa multistage microfluidic device that uses multiple regions to isolatetarget analytes that express different levels of magnetic moment. Insome implementations, the clogging can be minimized by using a differentconfiguration of the high magnetic permeability material and lowmagnetic permeability material in the magnetizable layer.

For example, the magnetizable layer can be constructed to include an“edge” configuration as shown in FIG. 19C, where there is a singleinterface between the high magnetic permeability material and the lowmagnetic permeability material, in place of the “gap” configuration. Incontrast to the gap configuration, in which magnetic particles (ortarget analytes bound to magnetic particles) are pulled toward regionsof the channel directly above the two interfaces between the lowmagnetic permeability material and the high magnetic permeabilitymaterial, the magnetic particles (or target analytes bound to magneticparticles) are pulled toward a single region above the single interfacebetween the low magnetic permeability region and the high magneticpermeability material in the edge configuration. The edge configurationthus has an advantage that target analytes are deflected toward a singleline, e.g., in or towards the middle of a microfluidic channel, insteadof being pulled to separate regions of the microfluidic channel, e.g.,towards the walls of the microfluidic channel), thereby improving thecollection and isolation of the target analytes.

FIG. 11 is a schematic of an example of an arrangement for a multistagedevice that utilizes high magnetic flux gradients to isolate targetanalytes. The multistage device includes a first stage 1102 forisolating analytes that exhibit high magnetic moments or are bound toparticles that exhibit high magnetic moments, a second stage 1104 forisolating analytes that exhibit relatively lower magnetic moments or arebound to particles that exhibit relatively lower magnetic moments, and athird stage 1106 for removing waste or other desired analytes. As shownin the schematic of FIG. 11, the first stage 1102 isolates the particlesexhibiting high magnetic moments by deflecting those particles toward afirst outlet 1103. The second stage 1104 isolates the particlesexhibiting relatively lower magnetic moments by deflecting thoseparticles toward a second outlet 1105.

Multi-stage devices, such as the two-stage device 1100, enable ahigh-dynamic range for isolating different analytes. For example, thefirst stage 1102 captures analytes that may have a large magnetic momentand that would otherwise be trapped in the second stage 1104. The largemagnetic moment may be due to a large number of magnetic particles boundto the analytes in a fluid sample or because the magnetic particles inthe fluid sample each have a high magnetic moment. The second stage 1104is more sensitive and thus can capture analytes that have a smallermagnetic moment. For example, the analytes captured in the second stagemay be bound to a second type of magnetic particles, each of which has alower magnetic moment relative to a first type of magnetic particle, orthe analytes captured in the second stage 1104 may be bound to fewermagnetic particles than the analytes captured in the first stage 1102.For example, if the analytes are cells, the first stage can capturecells that express a specific surface marker molecule at a high level(so that many magnetic particles are bound to the many surface markers),while the second stage can capture cells that express the same surfacemarker, but at a lower level (so that fewer magnetic particles are boundto the surface of these cells). The remaining analytes, such as whiteblood cells (WBC) in the example of FIG. 11, are delivered to the thirdstage 1106.

The multistage device shown in FIG. 11 addresses clogging by removing inthe first stage 1102 unbound magnetic particles and magnetic particleaggregates. With the reduction in clogging, the multi-stage deviceenables, in some implementations, the use of larger magnetic particlesand higher magnetic particle concentrations. In addition, the so-called“edge” configuration can also be used to avoid clogging by directing thedesired analytes to the center of the microfluidic channels, as opposedto the channel walls.

FIG. 12A is a schematic top view of an example of a system 1200 thatincludes two separate stages for analyte isolation based on deflectionby magnetic flux gradients. The first and second stages form a singlecontinuous channel having two approximately concentric loops. The system1200 includes an inlet 1206, a first focusing stage 1208, a firstdeflection channel/isolation stage 1202, and an outlet 1210 for one ormore first target analytes isolated in the first deflection channel1202. The deflection channel 1202 relies on a magnetizable layer thatincludes one or more low magnetic permeability regions arranged in ahigh magnetic permeability material to deflect the one or more firsttarget analytes. The deflection of the one or more first target analytescauses those analytes and some of the sample fluid to flow to outlet1210, where they are collected.

The system 1200 also includes a second inlet 1212 that receives aportion of the remaining fluid sample from the first deflection channel1202. The portion flowing into inlet 1212 may include all the remainingfluid sample that did not flow into outlet 1210. Alternatively, some ofthe remaining fluid sample from the first channel 1202 may be “waste”fluid and flow into outlet 1203, whereas a different portion of theremaining fluid sample containing second target analytes may flow intoinlet 1212. Once having passed second inlet 1212, the remaining fluidportion enters a second focusing stage 1214 and then a second deflectionchannel/isolation stage 1204, where the second target analytes aredeflected by the magnetic flux gradients into an outlet 1216 to becollected. Any waste fluid (i.e., the portion of the fluid sample lessthe first and second target analytes) in the second deflection channel1204 flows into outlet 1218.

The elongated gap region 1201 containing the low magnetic permeabilitymaterial extends across the length of the device substantiallyunderneath the second stage 1204. FIG. 12B is a schematic that shows aclose-up view of different sections of the first isolation stage 1202and the second isolation stage 1204. As shown in FIG. 12B, the elongatedgap region 1201 is arranged at an oblique angle with respect to acentral longitudinal axis of second isolation stage 1204. In contrast,no portion of the low magnetic permeability material 1201 extendsunderneath the adjacent first isolation stage 1202. As a result of thisconfiguration, the magnetic field gradient is much higher in secondstage 1204 than the field gradient in the first state 1202, and a muchgreater force may be exerted on magnetic particles in the secondisolation stage 1204 than in the first isolation stage 1202.

Thus, the second isolation stage 1204 may be better suited fordeflecting target analytes having a low overall magnetic moment (e.g.,analytes attached to small magnetic particles and/or particles havinglow magnetic moments), whereas the first isolation stage 1202 may bemore appropriate for deflecting target analytes expressing high magneticmoments (e.g., analytes attached to large magnetic particles and/orparticles having high magnetic moments) in which the magnetic force doesnot have to be very high to induce deflection.

In some implementations, clogging in a device, e.g., as those shown inFIG. 12, can be minimized by cycling the magnetic field on and off. Byremoving or turning off the magnetic flux source, the magnetic flux nearthe low magnetic permeability region is eliminated, allowing unboundmagnetic particles and magnetically labeled particles stuck to thechannel wall near the magnetizable layer to free themselves. For systemswith permanent magnets, cycling the magnetic field may includephysically separating the permanent magnet(s) from the device so thatthe field does not extend into the microfluidic channel. For systemswith electromagnets, cycling the magnetic field may entail cycling acurrent supplied to the electromagnet. By minimizing magnetic particleaccumulation, the device may be re-used, thus reducing waste.

Fabrication of Microfluidic Device

In general, a microfluidic device for isolating and/or separating targetanalytes using high magnetic flux gradients can be fabricated asfollows. Referring to FIG. 13, one or more magnets are initiallyprovided (1302). The magnet can be made of any suitable magneticmaterial capable of emitting a high magnetic field (e.g., alloys ofNdFeB, SmCo, AlNiCo, or ferrite). An optional spacer layer may then beprovided (1304) on a surface of the one or more magnets. The optionalspacer layer can include any suitable low magnetic permeabilitymaterial, e.g., a non-magnetic material, including, for example, glass,plastic or silicon.

A magnetizable layer then is formed (1306) adjacent to the one or moremagnets. For example, the magnetizable layer may be formed on a surfaceof the one or more magnets or on a surface of the optional spacer layer.The magnetizable layer may include, for example, a piece of adhesivemagnetic tape. Alternatively, the magnetizable layer may be deposited asa thin or thick film of magnetic material using any suitable depositiontechnique such as thermal deposition, plasma deposition,electro-plating, or electron-beam deposition. Preferably, themagnetizable layer includes a high magnetic permeability material havinghigh saturation flux density. As explained above, the greater thesaturation flux density, the greater the amount of flux that can passthrough the high magnetic permeability material leading to gains in fluxgradient. Materials that can be used for the high magnetic permeabilitymaterial include, but are not limited to, iron, nickel, cobalt, ornickel-iron alloys such as Ni₈₀Fe₂₀ or Ni₄₅Fe₅₅, steel, CoFeNi, FeAlNalloys, SiFe alloys, or CoFe alloys. In some implementations, the highmagnetic permeability material can be a composite material, such as apolymer, glass, or ceramic that contains high magnetic permeabilityparticles (e.g., iron, nickel, cobalt, or nickel-iron alloys such asNi₈₀Fe₂₀ or Ni₄₅Fe₅₅, steel, CoFeNi, CoFe alloy, FeAlN alloy, or SiFealloy, particles)

Forming the magnetizable layer can include forming a low magneticpermeability region in the magnetic material of the magnetizable layer.For example, when using adhesive magnetic tape as the magnetic material,one or more elongated portions of the magnetic tape can be cut out priorto placing the magnetic tape on the surface of the magnet or spacerlayer. Optionally, the elongated region may be formed by machining theadhesive magnetic tape to remove the magnetic material. Machining mayinclude any standard machining techniques such as turning, boring,drilling, milling, broaching, sawing, shaping, planing, reaming,tapping, grinding, electrical discharge machining, electrochemicalmachining, electron beam machining, photochemical machining, lasermilling, or ultrasonic machining. The cut out portion may be left emptyor can be filled with a low magnetic permeability material, e.g., anon-magnetic material, to form the low magnetic permeability region inthe magnetizable layer. In case the magnetic material is deposited asopposed to being an adhesive, the elongated region can be formed usingapplicable etching techniques such as wet etching or dry etching (e.g.,plasma etching). The thickness of the portion removed from the magneticmaterial in any case (machining, cutting or etching) may be equal to orless than a thickness of the magnetic material.

The cut out portion may be filled with a low magnetic permeabilitymaterial, e.g., a non-magnetic material, using techniques such asthermal deposition, plasma deposition, electro-plating, or electron-beamdeposition. An optional thin film layer (e.g., SiO₂) can be formed on asurface of the magnetizable layer using, for example, thermal orelectron beam deposition, such that the thin-film is conserved a part ofthe magnetizable layer.

In some implementations, the high magnetic permeability material can beformed using a molding process or a thermoforming process. For example,composite materials such as plastics, glass, or ceramics containingmagnetic particles, are amenable to molding or thermoforming processes.

After forming the magnetizable layer, the microfluidic channel and coverare formed above the magnetizable layer (1308). In some implementations,the microfluidic channel and cover are formed by depositing a polymer(e.g., PDMS, PMMA or polycarbonate (PC)) in a mold that defines thefluidic channel regions. The polymer, once cured, then is transferredand bonded to a surface of the magnetizable layer. For example, PDMS canbe first poured into a mold (e.g., an SU-8 mold fabricated with two stepphotolithography (MicroChem)) that defines the microfluidic network ofchannels. The PDMS then is cured (e.g., heating at 65° C. for about 3hours). Prior to transferring the solid PDMS structure 710 to thedevice, the surface of the oxide layers is treated with O₂ plasma toenhance bonding.

In some implementations, the microfluidic device can be fabricated suchthat it includes a removable and/or replaceable portion. The replaceableportion could include, for example, the microfluidic channel, such thatafter using the device one or more times (e.g., flowing a sample fluidthrough the microfluidic channel), the fouled channel can be disposed.The channel can then be replaced with a new fresh channel, thuseliminating a washing step and, in some implementations, leading to areduction in processing time. In some cases, designing the device toinclude the removable portion may also reduce fabrication costs.

FIG. 14A is a schematic of a first configuration 1500 of a microfluidicdevice that includes a removable and/or replaceable portion. Theconfiguration 1500 includes a cartridge portion 1502 and an instrumentportion 1504. The instrument portion includes the magnets 1506 forgenerating the magnetic field. The cartridge portion 1502 can beremovably fixed to the instrument portion 1504 and includes themicrofluidic channel cover 1508, the microfluidic channel 1510 (e.g.,which is defined by the microfluidic channel cover 1508), a passivationlayer 1512, which acts as the floor of the microfluidic channel 1510,the low permeability region 1514 (e.g., a gap containing vacuum or airor other low magnetic permeability material), and the high permeabilityregion 1516 (e.g., a magnetizable alloy).

FIG. 14B is a schematic of a second configuration 1550 of a microfluidicdevice that includes a removable and/or replaceable portion. Theconfiguration 1550 includes cartridge portion 1552 and an instrumentportion 1554. The instrument portion 1554 includes the magnets 1506 forgenerating the magnetic field, the low permeability region 1514 (e.g., agap containing vacuum or air or other low magnetic permeabilitymaterial), and the high permeability region 1516 (e.g., a magnetizablealloy). The cartridge portion 1552 can be removably fixed to theinstrument portion 1504 and includes the microfluidic channel cover1508, the microfluidic channel 1510 (e.g., which is defined by themicrofluidic channel cover 1508), and a passivation layer 1512, whichacts as the floor of the microfluidic channel 1510.

In either design, the passivation layer 1512 is preferablynon-magnetizable and is thin enough to minimize the distance between atop surface of the magnetizable alloy 1516 and the floor of themicrofluidic channel 1510. For example, the thickness of the passivationlayer could be less than approximately 5 μm. The passivation layer 1512can be bonded to the microfluidic cover 1508 using plasma or using anadhesive, such as epoxy. To ensure proper alignment, it is preferablethat the base layer does not stretch significantly. Examples ofmaterials for the passivation layer include coextruded metal and polymer(e.g., an aluminum layer sandwiched between polymer layers.).

To aid in coupling the cartridge portion 1502 (1552) to the instrumentportion 1504 (1554), the instrument portion 1504 (1554) and thecartridge portion 1502 (1552) can include alignment markers, such ascross-hairs or other shapes, which can be used to align the two portionsto one another. For example, for configuration 1500, the alignmentmarkers can be formed on the magnets 1506 and on one of the layers ofthe cartridge portion 1502 (e.g., the microfluidic channel cover 1508)using known etching or deposition techniques. In some implementations,the low permeability region 1514 itself can be used as an alignmentmarker. Alignment can be performed manually or using an automatedalignment system. The alignment marks can be used, in some cases, toenable alignment within 5 μm precision.

Once aligned, the cartridge portion 1502 (1552) can be fixed to theinstrument portion 1504 (1554). To enable the cartridge portion 1502(1552) to be removably fixed to the instrument portion 1504 (1554), thecartridge portion 1502 (1552) and instrument portion 1504 (1554) caninclude grooves or ridges that mate with one another and snap-lock intoplace. Alternatively, in some implementations, the cartridge portion1502 (1552) may include a ridge portion on its bottom surface thatslides into a groove formed on the instrument portion 1504 (1554) andlocks into place or vice versa. For example, the ridges may be formed tohave a T-shape (e.g., a wide top and narrow base) that slides into aslot formed on the instrument portion, where the slot has a wide openingat its base and narrow opening at the top to secure the cartridge inplace.

In some implementations, the layer containing the high magneticpermeability material and the low magnetic permeability material can beincluded as part of the cartridge portion 1552, but also be reusable.However, the microfluidic channel cover 1508 and passivation layer 1512would be disposable. In this example, the passivation layer would bereversibly bound or mechanically held to the layer containing the highmagnetic permeability material and the low magnetic permeabilitymaterial. After use of the device, the fluidic layer could beremoved/released from layer containing the high magnetic permeabilitymaterial and the low magnetic permeability material. In some cases, thealloy/gap part could then be returned, for example, to a source factoryto be reused with a new fluidic layer. An advantage of this approachwould be that the alignment of the fluidic layer with the layercontaining the high and low magnetic permeability materials could bedone in a central facility, rather than with the end user.

In some implementations, the orientation of the microfluidic channel(s)can be modified manually with respect to the magnetizable layer. Forexample, in some cases, the microfluidic channel is formed as part of acartridge that removably couples to an instrument portion containing themagnetizable layer and the one or more magnets. The cartridge may berotatably fixed or translated with respect to the magnetizable layer ofthe instrument portion such that the direction of the force induced bythe magnetic gradient changes relative to the flow direction of themicrofluidic channel. That is, the cartridge may be rotated ortranslated to one of several different positions with respect to themagnetizable layer and then fixed in place in any one of the differentpositions using a locking mechanism, such as, e.g., a combination ofridges and slots configured to mate with one another.

FIGS. 28A-28F are schematics depicting top views of examples ofdifferent arrangements of a microfluidic channel with respect to a highmagnetic flux gradient inducing structure. Each figure shows an outlineof a fluidic channel 2800 and a low magnetic permeability region 2802such as is formed in the “gap” structure of FIG. 1A. Alternatively, theregion 2802 could correspond to the interface between a high magneticpermeability material and the low magnetic permeability material in the“edge” configurations of the microfluidic device. Each fluidic channelincludes three possible outlets (“Output 1,” “Output 2,” and “Output3”). When the cartridge containing the fluidic channels is rotated (seeFIGS. 28A-28C) relative to the low magnetic permeability region 2803,the force experienced by a magnetic particle 2804 may cause the particle2804 to follow a trajectory toward one of the three outlets, dependingon the amount of rotation of the cartridge. For example, in FIG. 28A,the particle 2804 follows the low magnetic permeability region 2802toward output 1.

In FIGS. 28B and 28C, the particle 2804 follows the low magneticpermeability region 2802 toward outputs 2 and 3, respectively, as thefluidic channel is rotated. Alternatively, or in addition, the cartridgemay be translated relative to the low magnetic permeability region 2802(see FIGS. 28D-28F), a particle 2804 flowing through the fluidic channel2800 again may experience a force that causes it to flow toward one ofthe different output, depending on the amount of shift. For example, inFIG. 28D, the fluidic channel is shifted downward with respect to theregion 2802, such that the particle 2804 follows the low magneticpermeability region 2802 toward output 1, whereas in FIGS. 28E and 28F,the particle 2804 follows the low magnetic permeability region 2802toward outputs 2 and 3, respectively.

Microfluidics

In some implementations, the microfluidic channel 112 of themicrofluidic devices described herein can be a part of a larger optionalmicrofluidic channel network. Such microfluidic networks can be used tofacilitate control and manipulation (e.g., separation, segregation) ofsmall volumes of liquid and help isolate target analytes from a complexparent specimen. During the isolation process, microfluidic elementsprovide vital functions, for example, handling of biological fluids,reproducible mixing of magnetic particles with samples, distribution ofaliquots to different coils for parallel sensing, and confining ofsamples to the most sensitive region of a given microcoil. Additionalinformation about microfluidic channel networks and their fabricationcan be found in U.S. Patent App. Publication No. 2011/0091987, e.g., inparagraphs eighty-one to eighty-eight.

Use of Magnetic Particles

As noted above, a fluid sample that may contain a target analyte that ismixed with a liquid containing a number of particles that are designedto specifically bind to the target analyte. The particles can includemagnetic particles (e.g., nanoparticles) that form a target-particlecomplex in solution.

Particles

Magnetic particles can include one or more inner magnetic cores and anouter coating, e.g., a capping polymer. The magnetic cores can bemonometallic (e.g., Fe, Ni, Co), bimetallic (e.g., FePt, SmCo, FePd,FeAu) or can be made of ferrites (e.g., Fe₂O₃, Fe₃O₄, MnFe₂O₄, NiFe₂O₄,CoFe₂O₄). The magnetic particles can be nanometers or micrometers insize, and can be diamagnetic, ferromagnetic, paramagnetic, orsuperparamagnetic, in which size corresponds to an average diameter oraverage length. For example, the magnetic particles can have a size ofapproximately 1 μm, approximately 600 nm, approximately 500 nm,approximately 300 nm, approximately 280 nm, approximately 160 nm, orapproximately 100 nm. Other particle sizes are possible as well. Theouter coating of a particle can increase its water-solubility andstability and also can provide sites for further surface treatment withbinding moieties.

Binding Moieties

In general, a binding moiety is a molecule, synthetic or natural, thatspecifically binds or otherwise links to, e.g., covalently ornon-covalently binds to or hybridizes with, a target molecule, or withanother binding moiety (or, in certain embodiments, with an aggregationinducing molecule). For example, the binding moiety can be a syntheticoligonucleotide that hybridizes to a specific complementary nucleic acidtarget. The binding moiety can also be an antibody directed toward anantigen or any protein-protein interaction. Also, the binding moiety canbe a polysaccharide that binds to a corresponding target. In certainembodiments, the binding moieties can be designed or selected to serve,when bound to another binding moiety, as substrates for a targetmolecule such as enzyme in solution. Binding moieties include, forexample, oligonucleotides, polypeptides, antibodies, andpolysaccharides. As an example, streptavidin has four sites (bindingmoieties) per molecule that will be recognized by biotin. For any givenanalyte, e.g., a specific type of cell having a specific surface marker,there are typically many known binding moieties that are known to thoseof skill in the relevant fields.

For example, certain labeling methods and binding moiety techniques arediscussed in detail in U.S. Pat. No. 6,540,896 entitled,“Microfabricated Cell Sorter for Chemical and Biological Materials”filed on May 21, 1999; U.S. Pat. No. 5,968,82050 entitled, “Method forMagnetically Separating Cells into Fractionated Flow Streams” filed onFeb. 26, 1997; and U.S. Pat. No. 6,767,706 entitled, “Integrated ActiveFlux Microfluidic Devices and Methods” filed Jun. 5, 2001.

Conjugate Preparation

The surface of the magnetic particles are treated to present functionalgroups (e.g., —NH₂, —COOH, —HS, —C_(n)H_(2n-2)) that can be used aslinkers to subsequently attach the magnetic particles to cells othertarget molecules (e.g., antibodies, drugs). In some cases, the surfacetreatment makes the magnetic particles essentially hydrophilic orhydrophobic. The surface treatment can be formed of polymers including,but not limited to, synthetic polymers such as polyethylene glycol orsilane, natural polymers, derivatives of either synthetic or naturalpolymers, and combinations thereof.

In some implementations, the surface treatment is not a continuous filmaround the magnetic particle, but is a “mesh” or “cloud” of extendedpolymer chains attached to and surrounding the magnetic particle.Exemplary polymers include, but are not limited to polysaccharides andderivatives, such as dextran, pullanan, carboxydextran, carboxmethyldextran, and/or reduced carboxymethyl dextran, PMMA polymers andpolyvinyl alcohol polymers. In some implementations, these polymercoatings provide a surface to which targeting moieties and/or bindinggroups can bind much easier than to the shell material. For example, insome embodiments magnetic particles (e.g., iron oxide nanoparticles) arecovered with a layer of 10 kDa dextran and then cross-linked withepichlorohydrin to stabilize the coating and form cross-linked magneticparticles.

Additional information on the fabrication, modification and use ofmagnetic particles can be found, for example, in PCT Pub. No.WO/2000/061191, U.S. Patent App. Pub. No. 20030124194, U.S. Patent App.Pub. No. 20030092029, and U.S. Patent App. Pub. No. 20060269965.

Applications

The new microfluidic device described herein can be used in variousapplications including, for example, as part of a research platform tostudy analytes of interest (e.g., proteins, cells (such as circulatingtumor cells (CTCs) or fetal cells, e.g., in maternal blood), bacteria,pathogens, and DNA) or as part of a diagnostic assay for diagnosingpotential disease states or infectious agents in a patient. Examples ofdetection targets are discussed in more detail below and in the Examplessection.

Detecting Infectious Agents

By modifying the functional ligands (e.g., binding moieties) on themagnetic particles, the microfluidic devices described herein can beused to detect, isolate, and/or measure many different biologicalanalytes, including small molecules, proteins, nucleic acids, pathogens,and cells, e.g., rare cells such as cancer cells.

Rare Cell Detection

The microfluidic devices and methods described herein can be used todetect rare cells, such as circulating tumor cells (CTC) in a bloodsample, or fetal cells in blood samples of pregnant females. Forexample, primary tumor cells or CTCs can be targeted and linked tomagnetic particles and can be detected using the new microfluidic devicefor a rapid and comprehensive profiling of cancers. By changing bindingmolecules on the magnetic particle surface, different types of cells canbe detected (e.g., circulating endothelial cells for heart disease).Thus, the microfluidic device may be used as a powerful diagnostic andprognostic tool. The targeted and detected cells can be cancer cells,stem cells, immune cells, white blood cells, or other cells including,for example, circulating endothelial cells (using an antibody to anepithelial cell surface marker, e.g., the Epithelial Cell AdhesionMolecule (EpCAM)), or circulating tumor cells (using an antibody to acancer cell surface marker, e.g., the Melanoma Cell Adhesion molecule(CD146)). In some implementations, the system sensitivity can detect aslow as a few cells or less per milliliter of detection volume, i.e., thedevice itself has the capacity for single-cell detection. The systemsand methods also can be used to detect small molecules, proteins,nucleic acids, or pathogens.

Multiplexed Detection

Detecting multiple biomarkers in one parent sample is an important andhighly desirable task for diagnosis and prognosis of complex diseases.For example, there is no ubiquitous biomarker for cancer;multi-channeled screening is required to correctly identify tumor types.The new microfluidic devices described herein offer methods to detectdifferent relevant biomarkers from the aliquots of a single, parentsample, e.g., in patients with cancer or metabolic disorders. Amultistage microfluidic device is well suited for this application. In amultistage device, different target analytes (e.g., white blood cellversus red blood cell) may be bound to different magnetic particles ordifferent amounts of magnetic particles such that the different targetanalytes exhibit a different response to the flux gradient in themicrofluidic channel. The target analytes bound to magnetic particleshaving a high responsivity may be deflected easier than target analytesbound to magnetic particles having a lower responsivity.

Thus, in a first stage of the microfluidic device, the analytesexpressing higher responsivity may be filtered out of the sample toisolate the analytes expressing the lower responsivity (or vice versa).In a second stage of the microfluidic device, the analytes expressingthe lower responsivity to the flux gradient then can be filtered outfrom the sample. Examples of tumor cell biomarkers that can be detectedinclude MUC-1, EGFR, B7-H3, Her2, Ki-67, EpCam, Vim, and CK18.

EXAMPLES

The invention is further described in the following example, which doesnot limit

the scope of the invention described in the claims.

Example 1—Isolating Cancer Cells in Blood

The purpose of this example was to simulate a cancer patient's blood byspiking cell line cancer cells (CTCs) into a healthy donor's bloodsample, efficiently tagging these cancer cells with magnetic beads, andisolating the cancer cells for detection and/or further measurements.White blood cells also were analyzed.

Device Fabrication

Several different types of microfluidic devices were fabricated andevaluated to determine performance. To fabricate the devices, thefabrication steps outline above with respect to FIG. 13 were followed.In particular, a 1 mm thick glass substrate was provided for eachdevice. Approximately 1 μm of FeAlN was deposited on the surface of theglass substrates using sputtering. The saturation flux density of FeAlNis about 1.8 T. The FeAlN layer was then patterned into multipleparallel arrangements of stripes. Patterning was accomplished bysputtering FeAlN onto the glass substrate and then etching FeAlN awayfrom areas outside the stripes. Each sample had a different stripe widthand/or separation distance (gap) between stripes.

Table 1 below provides the dimensions for the different sample devicesM5.1 to M5.5. The width of the low magnetic permeability region for the“gap” device was 20 μm.

TABLE 1 Stripe width Gap width Design (μm) (μm) M5.1 20 10 M5.2 40 40M5.3 40 5 M5.4 40 20 M5.5 60 20

The surfaces of the stripes were then passivated with a thin layer ofSiO₂ (approximately 0.2 μm thick). The microfluidic channels werefabricated using standard soft lithography. A SU-8 (MicroChem) mold wasfabricated using photolithography. The shape of the mold defined amicrofluidic channel region. Polydimethylsiloxane (PDMS) was poured ontothe mold and cured at 65° C. for about 8 hours. For each device, thePDMS microfluidic cover and was treated with O₂ plasma, aligned with thedevice substrates using a mask aligner, and permanently bonded to thesubstrates. The PDMS cover was aligned with the stripes such that thestripes were at an approximately 1° angle from a central longitudinalaxis of the microfluidic channel. The “gap” device was constructed withan angle of about 0.5° from a central longitudinal axis of themicrofluidic channel.

Magnetic Bead Preparation

Magnetic beads having a diameter of about 1.0 μm were prepared for usein whole blood for labeling CTCs.

In particular, streptavidin coated magnetic beads (Dynabeads MyOneStreptavidin T1 magnetic beads from Invitrogen) were incubated withbiotinylated anti-hEpCAM antibodies (R&D Biosystems). Anti-hEpCAM coatedbeads bind specifically to CTCs that express EpCAM molecules on theirsurface. The 1 μm magnetic beads used in this procedure were washed with0.01% TWEEN® 20 (Fisher Scientific) diluted in 1×PBS. EpCAM antibody wasthen added to the beads, which were washed again by using 0.01% TWEEN 20diluted in 1×PBS. During antibody incubation, antibody concentration wasabout 100 μg/ml and bead concentration was about 2 mg/ml. Theconcentration was then increased to about 5 mg/ml by reducing the volumeright before being spiked into the blood sample. 160 nm magnetic beads(Veridex Ferrofluid) were received from Veridex ready to use (i.e., thebeads are pre-functionalized with EpCAM ourselves).

Sample Preparation

EpCAM were spiked into a healthy whole blood fluid sample, and then 1.0μm magnetic beads coated with EpCAM antibody or the 160 nm magneticbeads coated with EpCAM antibody were added to the fluid sample. Thesample was added to a sample tube and actively mixed using a tri-polemagnet (Vendex) so as to bind the magnetic beads to the CTCs. Activemixing included placing the sample tube adjacent to the magnet andmodifying the tube orientation over a period of several minutes. Activemagnetic mixing increases the number of collisions between the beads andtarget cells and enhances the chance of interaction.

Device Operation

1% Pluronic F68 (BASF) was first prepared and introduced into themicrofluidic channel of the microfluidic device for about 15 minutesusing a syringe pump (New Era Pump System) to prime the microfluidicdevice. After the buffer solution was collected at an outlet of themicrofluidic device, the blood sample fluid (approximately 7 mL)containing the mixture of CTCs bound to magnetic beads was introducedinto the microfluidic device. Prior to introducing the blood sample intothe microfluidic device, the blood was debulked (i.e., RBCs, free beads,platelets were removed). The microfluidic device isolated magneticallylabeled cells within the sample, from non-labeled cells. The portion ofthe fluid sample containing the magnetically labeled cells werecollected in a product tube and the remaining blood sample was collectedin a waste tube. After the blood sample passed through the microfluidicdevice, the channel was once again flushed with buffer solution.

The blood sample collected in the product tube and the waste sample wereanalyzed to get an accurate count of spiked cell and WBC contents inthem. Spiked cells were pre-stained with CELLTRACKER® Red to allow themanual count from the IFD product and waste samples. The blood samplealso was stained with Calcein-AM, thus staining both the WBCs andspecific cells, and allowing a count of WBC concentration as well.

Spike cell and WBC concentrations in the blood sample and waste weremeasured or found by manual counting; then, the total number of cells ineach of them was interpolated to estimate percent recovery yields andproduct purity.

Results

The metrics of device performance are (1) relative yield, (2) absoluteyield, and (3) white blood cell carryover (or purity). The relativeyield is defined as the percentage of counted output (product+waste)cells found in the product. The absolute yield is defined as thepercentage of (assumed) input (spiked) cells found in the product.

Tables 2, 3, and 4 summarize the results of magnetic design comparisonexperiments. Table 2 shows relative yield, Table 3 shows absolute yield,and Table 4 shows white blood cell carryover. In the Bead Type column,“DB” indicates Dynal Dynabeads (1 μm diameter) and “FF” indicatesVeridex Ferrofluid (160 nm diameter).

TABLE 2 Relative Yield in Comparison Experiments Experimental ConditionsBead Conc. Relative Yield (%) Exp. Cell Bead (μg/ Stripe Gap # Line TypemL) M5.1 M5.2 M5.3 M5.4 M5.5 M6 1 MB231 DB 100 83 2 SKBR3 FF 100 97 3PC3-9 FF 4.4 62 4 PC3-9 FF 4.4 81 5 PC3-9 FF 22 68 67 64 6 PC3-9 FF 4.413 11 15 7 PC3-9 DB 30 96 98 97 8 PC3-9 DB 6 31 31 20 9 PC3-9 DB 15 6785 54 10 PC3-9 FF 22 30 11 SKBR3 DB 30 100 100 95 12 PC3-9 FF 4.4 26 5713 PC3-9 FF 4.4 10 89 14 PC3-9 FF 4.4 79 88 15 MB231 FF 4.4 42 76

TABLE 3 Absolute Yield in Comparison Experiments Experimental ConditionsBead Conc. Absolute Yield (%) Exp. Cell Bead (μg/ Stripe Gap # Line TypemL) M5.1 M5.2 M5.3 M5.4 M5.5 M6 1 MB231 DB 100 70 2 SKBR3 FF 100 77 3PC3-9 FF 4.4 60 4 PC3-9 FF 4.4 47 5 PC3-9 FF 22 66 71 62 6 PC3-9 FF 4.49 7 13 7 PC3-9 DB 30 86 68 60 8 PC3-9 DB 6 33 30 24 9 PC3-9 DB 15 72 8253 10 PC3-9 FF 22 25 11 SKBR3 DB 30 36 32 47 12 PC3-9 FF 4.4 30 54 13PC3-9 FF 4.4 11 60 14 PC3-9 FF 4.4 64 66 15 MB231 FF 4.4 37 71

TABLE 4 WBC Carryover in Comparison Experiments Experimental ConditionsBead WBC Carryover (WBCs/mL Blood) Bead Conc. Stripe Gap Exp. # CellLine Type (μg/mL) M5.1 M5.2 M5.3 M5.4 M5.5 M6 1 MB231 DB 100 5404 2SKBR3 FF 100 1602 3 PC3-9 FF 4.4 1213 4 PC3-9 FF 4.4 1665 5 PC3-9 FF 222621 4501 5600  6 PC3-9 FF 4.4  364 397 484* 7 PC3-9 DB 30  838 18761109  8 PC3-9 DB 6   96* 27 16 9 PC3-9 DB 15 2188 3358 509 10 PC3-9 FF22  669 11 SKBR3 DB 30 6661 3340 448  12 PC3-9 FF 4.4  379 537 13 PC3-9FF 4.4 83 176 14 PC3-9 FF 4.4  281* 306

As can be seen from Table 2, each of the different devices had very highrelative yield for most of the different concentrations of beads andbead types. In general, the “gap” device was equivalent to orsubstantially outperformed the “stripe” devices with respect to relativeyield and absolute yield. As shown in Table 4, the “gap” device also wasable to achieve a higher white blood cell carryover than the “stripe”devices, thus indicating that a gap design may provide the highestsensitivity for isolating magnetically labeled analytes in a fluidsample.

FIGS. 15A and 15B are bar graphs that show the bead load required forcapture in a Stripe device fabricated according to M5.2 in Table 1.Here, the bead load is quantified in terms of the number of Dynal MyOneDynabeads (1 μm diameter) counted on MB321 cells in the blood sample andwaste collected in the experiment. If a threshold bead load for captureis defined as the bead load at which 50% of the target cells arecaptured, then the threshold bead load for the stripe device is a low 3beads. Accordingly, the stripe design is extremely sensitive to smalllevels of magnetic moment and provides an effective approach toisolating analytes in a fluid sample.

Example 2—Simulations of Field Gradient in Two-Stage Devices

Various simulations were also performed to analyze the operation of anintegrated microfluidic device constructed according to the presentdisclosure. For example, FIG. 16 includes a schematic cross-section ofan integrated microfluidic device 1600 used in simulations of magneticfield gradients. The device 1600 is based on the configuration shown inFIG. 12, in which a fluidic layer 1610 having two target isolationstages is arranged above a magnetizable layer 1604 (“Alloy (base) layer”in the figure) containing a high magnetic permeability portion 1606 anda low magnetic permeability portion 1608. Two magnets 1602 are arrangedbeneath the magnetizable layer 1604 to provide the magnetic field. Thesecond isolation stage is a fluidic channel positioned directly abovethe elongated gap region comprising the low magnetic permeabilityregion. The first isolation stage also is a fluidic channel but islaterally offset from the second isolation stage and the low magneticpermeability region. FIG. 16 also includes a heat map illustrating asimulated magnetic field gradient corresponding to a section of theintegrated microfluidic device 1600 that includes a portion of themagnets 1602, the magnetizable layer 1604 and the two isolation stages.The region of the heat map containing the two isolation stages isenlarged for ease of viewing the field gradients.

The simulations were performed using COMSOL finite element analysissoftware. The permanent magnets 1602 were assumed to be 5 mm×5 mm with1.3 T remnant magnetization and having the polarity as shown in FIG. 16.The high magnetic permeability material was 500 μm thick and had 1.8 Tsaturation flux density. The high magnetic permeability materialextended 6 mm to the left of the low magnetic permeability material inthe gap and 20 mm to the right of the gap. The gap width was 40 μm. Thesystem boundaries were far from the device, with approximately 100mm×100 mm overall dimensions. The relative permeability (the ratio ofthe permeability of the medium to that of free space permeability) ofthe high magnetic permeability material was assumed to be 10,000 and therelative permeability of the low magnetic permeability material wasassumed to be 1.

As shown in the heat map, there are both local minima 1620 and localmaxima 1630 in the field gradient. For this “gap” structure, in whichthe low magnetic permeability material is positioned between the highmagnetic permeability material, two local maxima 1630 occur within thesecond isolation stage (“Stage 2”) itself. In contrast, given thelateral displacement of the first isolation stage (“Stage 1”) from thegap region, the field gradient is fairly uniform across the firstisolation stage at a magnitude lower than the maxima in the secondisolation stage. As a result, a magnetic particle in the secondisolation stage will experience a greater deflection force than the sameparticle in the first isolation stage. The additional force may beuseful for isolating target analytes that express a low overall magneticpermeability (e.g., low magnetic bead load and/or small magnetic beadsize). In contrast, the first isolation stage may be used for isolatingtarget analytes that express a higher relative overall magneticpermeability (e.g., high magnetic bead load and/or large magnetic beadsize).

For example, FIG. 17 is a heat map showing a view of the magnetic fieldgradient in the first isolation stage. The different sections (1-5) ofthe heat map are cross-sectional views of the gradient in the x-y planeand correspond to different positions along the fluid flow direction, asshown in the top view of the first isolation stage on the left of theheat map. Top view images of the analytes in a fluid sample at thedifferent sections are superimposed on the heat map. At the end of thefluid channel, the first isolation stage separates into three separatepathways: one for the desired product, one for waste material, and onefor the remaining fluid sample that contains other target analytes(“Stage 2”). Though not shown, the low magnetic permeability region ofthe magnetizable layer is located to the left of the first isolationstage (i.e., along the x-direction).

Thus, the strength of the magnetic force generated by the field gradientis slightly greater to the left of the channel shown in FIG. 17 than tothe right. Due to the slightly higher magnetic force, target analytesexpressing a high overall magnetic permeability will be deflected to theleft of the channel and toward the product pathway. For example, threedifferent analytes are shown superimposed over the heat map of FIG. 17:a first circulating tumor cell (CTC) that is bound to a large number ofmagnetic beads (a high expresser target analyte), a second CTC that isbound to a much smaller number of magnetic beads (a low expresser targetanalyte), and a white blood cell (WBC). Under fluid flow, the highexpresser target is more sensitive to the slightly greater magneticforce and is deflected to the left of the channel, whereas both the lowexpresser target analyte and the WBC remain analyte are less sensitiveand remain traveling along the channel with the fluid flow towards Stage2.

FIG. 18 is a heat map showing a view of the magnetic field gradient inthe second isolation stage. Again, the different sections (1-5) of theheat map are cross-sectional views of the gradient in the x-y plane andcorrespond to different positions along the fluid flow direction, asshown in the top view of the second isolation stage on the left of theheat map. For the second isolation stage, however, the gap regioncontaining low magnetic permeability material is positioned underneaththe channel at an oblique angle with respect to the channel's centrallongitudinal axis. As shown in the heat map, maxima in the magneticgradient shift to the right of the channel as one moves along fromsection (1) to section (5). Given the high magnetic force, targetanalytes expressing a magnetic permeability will be deflected to theright of the channel. For example, the low expresser CTC will bedeflected toward the product pathway, whereas the WBC will follow thefluid flow toward the waste pathway.

Experimental tests using the two stage integrated microfluidic devicewere also conducted to observe how well the first stage and second stageisolate cells. In those experiments, both DYNABEADS® (approximately 1 μmin diameter) and Ferrofluid beads (approximately 160 nm in diameter)were bound to different cell lines (e.g., MB231, PC3-9, and SKBR3) andintroduced in a fluid sample into the two stage device. When the beadload was large (i.e., large bead and/or the cell line expressed a highmagnetic moment), most cells were captured by the less sensitive Stage 1(e.g., at least about 80% cell capture). However, when the bead load wassmall (i.e., small beads and/or the cell line expressed a low magneticmoment) most cells were captured by the more sensitive Stage 2.

Example 3—Simulations of Field Gradients for Different Device Parameters

FIGS. 20A and 20B include heat map plots of the simulated magnetic fieldgradient in the first and second isolation stages for both the gapconfiguration and the “edge” configuration shown in FIGS. 19B and 19C,respectively. In the edge configuration of FIG. 19C, a single localmaximum in the magnetic field gradient is produced in the channelcorresponding to the second isolation stage (see FIG. 20B), as opposedto two local maxima in the second isolation stage of the gap design (seeFIG. 20A). Thus, for the gap configuration of FIG. 19A, magneticparticles (or target analytes bound to magnetic particles) will tend tobe pulled toward regions of the channel directly above the two edgescorresponding to the two interfaces between the low magneticpermeability material and the high magnetic permeability material. Incontrast, in the edge configuration, the magnetic particles (or targetanayltes bound to magnetic particles) will be pulled toward a singleregion corresponding to the area above the single interface between thelow magnetic permeability region and the high magnetic permeabilitymaterial. The edge configuration thus has an advantage that targetanalytes are deflected toward a single line, instead of being pulled toseparate regions of the fluidic channel, improving the collection andisolation of the target analytes.

Various parameters of the integrated microfluidic device can be alteredto modify the gradient in magnetic flux and improve the deviceperformance. FIG. 22 is a schematic of a cross-section of the portion ofthe microfluidic device excluding the microfluidic channels and magnets.As can be seen from FIG. 22, some of the parameters that can be alteredto include the thickness 2202 a of the passivation/floor layer 2202beneath the microfluidic channels, the total thickness 2204 of thepassivation layer and the magnetizable layer (which includes the highmagnetic permeability layer 2206 and the low magnetic permeability gap2208 shown in FIG. 22), the thickness 2210 a of the low magneticpermeability material 2210 (i.e., the “plastic layer”) beneath the highmagnetic permeability material 2206, and the saturation flux density ofthe high magnetic permeability material.

To evaluate some of these parameters, several simulations were conductedfor the gap configuration shown in FIG. 21A and for the gapconfiguration shown in FIG. 21B. As indicated above, the simulationswere performed using COMSOL finite element analysis software. Thepermanent magnets were 5 mm×5 mm with 1.3 T remnant magnetization andhad a polarity as shown in FIG. 21A. The high magnetic permeabilitymaterial extended 6 mm to the left of the low magnetic permeability gapand 20 mm to the right of the gap. The gap width was 40 μm. The systemboundaries were far from the device, with approximately 100 mm×100 mmoverall dimensions. The relative permeability (the ratio of thepermeability of the medium to that of free space permeability) of thehigh magnetic permeability material was assumed to be 10,000 and therelative permeability of the low magnetic permeability material wasassumed to be 1. Only one parameter was varied at a time for eachsimulation.

FIG. 23A corresponds to the configuration in which the two fluidicchannels are spaced far apart (i.e., the configuration shown in FIG.21B). FIG. 23B corresponds to the configuration in which the fluidicchannels are spaced close together (i.e., the configuration shown inFIG. 21A). Each channel in both configurations was assumed to be 500 μmwide. Additionally, the low magnetic permeability gap region 2308 wasassumed to extend parallel to the central longitudinal axis of thesecond isolation stage “S2,” as opposed to an oblique angle to the axis.For the configuration shown in FIG. 23A, a first edge of the firstisolation stage “S1” was assumed to be laterally offset from the lowmagnetic permeability gap 2308 region by 1000 μm. However, for theconfiguration shown in FIG. 23B, the lateral offset of the edge of thefirst isolation stage from the low magnetic permeability gap 2308 was550 μm. For the purposes of the plots shown in FIGS. 24-26, theconfiguration shown in FIG. 23A is referred to as “IFD v.9.6/v.9.8,”whereas the configuration shown in FIG. 23B is referred to as “IFDv.9.7/v.9.9.”

FIG. 24A is a plot of the magnitude of the gradient in the simulatedmagnetic flux across the width fluidic channel in the first isolationstage (S1) as a function of saturation flux density of the high magneticpermeability material surrounding the gap region. As shown in the plot,the greater the saturation flux density of the high magneticpermeability material, the greater the magnetic force that can beapplied in both device configurations. Similarly, as shown in FIG. 24B,the average magnetic force in the second isolation stage (S2) alsoincreases proportionally to saturation flux density of the high magneticpermeability material. Accordingly, a microfluidic device manufacturedaccording to the present disclosure should preferably use the maximumsaturation flux density possible for the high magnetic permeabilitymaterial. For the simulations shown in FIGS. 24A and 24B, the total basethickness (including the floor thickness and the magnetizable layerthickness) was assumed to be 500 μm (i.e., the high magneticpermeability material thickness was fixed at 500 μm, the floor thicknesswas fixed at 0 μm, and the low magnetic permeability material thickness(i.e., the plastic layer) was fixed at 0 μm.

FIG. 25A is a plot of the magnitude of the gradient in the simulatedmagnetic flux across the width fluidic channel in the first isolationstage (S1) as a function of the passivation layer floor thickness. Ascan be seen in the plot, for both devices the magnetic force across thechannel is essentially constant until a floor thickness of about 100 μmis reached. At that point, the distance of the channel from the highmagnetic permeability material begins to reduce the size of the fieldgradient. The effect of increasing floor thickness is even moreprominent in the second isolation stage, as shown in FIG. 25B where thefield gradient decreases from about 14 kT/m at 0.1 micron to about lessthan 1 kT/m at 200 microns. Accordingly, a preferable passivation layerthickness should be less than about 10 μm, or as thin as fabricationprocesses will allow. The total base thickness (including the floorthickness and the magnetizable layer thickness) for each plot in FIG. 25was assumed to be 500 μm. The saturation flux was fixed at 1.8 T for thehigh magnetic permeability material. The high magnetic permeabilitymaterial thickness was fixed at 500 μm. The low magnetic permeabilitymaterial located beneath the high magnetic permeability material (i.e.,the plastic layer) had a thickness fixed at 0 μm.

FIG. 26A is a plot of the magnitude of the gradient in the simulatedmagnetic flux across the width fluidic channel in the first isolationstage (S1) as a function of the high magnetic permeability materialthickness for different total base thicknesses (i.e., the combinedthickness of the passivation layer, the high magnetic permeabilitymaterial, and the low magnetic permeability material, if any, beneaththe high magnetic permeability material). As shown in FIG. 26A, themagnetic force induced by the field gradients generally increases withincreasing thickness of the high magnetic permeability material (i.e.,the magnetic alloy). FIG. 26A also confirms that the gap configurationin which the two isolation stages are spaced closer together generallyperforms better for a particular base thickness than the gapconfiguration in which the isolation stages are separate further apartfor the same thickness.

FIG. 26B is a plot of the average magnitude (average over the 100 μm×50μm region centered over the gap) of the gradient in the simulatedmagnetic flux in the second isolation stage versus the alloy thickness.As shown in FIG. 26B, even though the magnetic force increases withalloy thickness similar to the first isolation stage, the magnetic forceplateaus at around 100-200 μm. Thus, a typical device should have athickness of at least about 200 μm to maximize the magnetic force in atleast the second isolation stage of the microfluidic device. For both ofthe plots produced in FIGS. 26A and 26B, the high magnetic permeabilitymaterial saturation flux was fixed at 1.8 T, the passivation floorthickness was fixed at 0 μm, and the base thickness fixed at either 500μm or 1000 μm, as indicated by legend. The low magnetic permeabilitythickness (i.e., the plastic layer thickness) was equal to thedifference between base thickness 500 μm (or 1000 μm) minus the highmagnetic permeability material thickness.

In general, an increase in total base thickness will reduce the magneticforce experienced in the first isolation stage for a fixed high magneticpermeability material thickness on the order of 0.1 to 0.25 T/m per 1 μmincrease in base thickness. For the second isolation stage, the magneticforce may experience a minimal increase with increasing base thickness.Accordingly, base thickness does not appear to be a critical parameterfor maximizing the magnetic force within the channels.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

What is claimed is:
 1. A microfluidic device comprising: a plurality ofmagnets, wherein each magnet of the plurality of magnets emits amagnetic field; a magnetizable layer arranged adjacent to the pluralityof magnets; and a microfluidic channel arranged adjacent to a surface ofthe magnetizable layer, wherein the magnetizable layer is configured toinduce a gradient in the magnetic field of at least one of the magnets,wherein the gradient extends into the microfluidic channel, wherein themagnetizable layer comprises a high magnetic permeability regioncomprising a composite material including a plurality of magneticparticles or comprising a high magnetic permeability material, and a lowmagnetic permeability region directly adjacent to the high magneticallypermeability region, wherein a central longitudinal axis of themicrofluidic channel extends substantially in parallel with an interfacebetween the high magnetic permeability region and the low magneticpermeability region, and wherein the magnetizable layer is configured tobe removably secured to the plurality of magnets or to the microfluidicchannel.
 2. The microfluidic device of claim 1, comprising a pluralityof high magnetic permeability regions and a plurality of low magneticpermeability regions arranged in an array that alternates between highand low magnetic permeability regions, wherein each high magneticpermeability region comprises the composite material.
 3. Themicrofluidic device of claim 1, wherein the high magnetic permeabilityregion comprises the composite material, and wherein the compositematerial comprises one or more of a polymeric material, a glassmaterial, or a ceramic material.
 4. The microfluidic device of claim 1,wherein a first magnet of the plurality of magnets is arranged above themagnetizable layer and a second magnet of the plurality of magnets isarranged below the magnetizable layer and facing the first magnet. 5.The microfluidic device of claim 4, wherein a side of the first magnetthat faces the second magnet has a first magnetic pole, and a side ofthe second magnet that faces the first magnet has a second magnetic polethat is opposite in polarity to the first magnetic pole.
 6. Themicrofluidic device of claim 1, wherein the low magnetic permeabilityregion has a relative magnetic permeability that is lower than arelative magnetic permeability of the high magnetic permeability regionby a factor of at least about
 4. 7. The microfluidic device of claim 1,wherein the microfluidic channel is rotatably fixed with respect to themagnetizable layer.